Plant starch composition

ABSTRACT

Disclosed is a nucleotide sequence encoding an effective portion of a class A starch branching enzyme (SBE) obtainable from potato plants, or a functional equivalent thereof, together with, inter alia, a corresponding polypeptide, a method of altering the characteristics of a plant, a plant having altered characteristics; and starch, particularly starch obtained from a potato plant, having novel properties.

FIELD OF THE INVENTION

This invention relates to novel nucleotide sequences, polypeptidesencoded thereby, vectors and host cells and host organisms comprisingone or more of the novel sequences, and to a method of altering one ormore characteristics of an organism. The invention also relates tostarch having novel properties and to uses thereof.

BACKGROUND OF THE INVENTION

Starch is the major form of carbon reserve in plants, constituting 50%or more of the dry weight of many storage organs—e.g. tubers, seeds ofcereals. Starch is used in numerous food and industrial applications. Inmany cases, however, it is necessary to modify the native starches, viachemical or physical means, in order to produce distinct properties tosuit particular applications. It would be highly desirable to be able toproduce starches with the required properties directly in the plant,thereby removing the need for additional modification. To achieve thisvia genetic engineering requires knowledge of the metabolic pathway ofstarch biosynthesis. This includes characterisation of genes and encodedgene products which catalyse the synthesis of starch. Knowledge aboutthe regulation of starch biosynthesis raises the possibility of“re-programming” biosynthetic pathways to create starches with novelproperties that could have new commercial applications.

The commercially useful properties of starch derive from the ability ofthe native granular form to swell and absorb water upon suitabletreatment. Usually heat is required to cause granules to swell in aprocess known as gelatinization, which has been defined (W A Atwell etal, Cereal Foods World 33, 306-311, 1988) as “ . . . the collapse(disruption) of molecular order within the starch granule manifested inirreversible changes in properties such as granular swelling; nativecrystallite melting, loss of birefringence, and starch solubilization.The point of initial gelaltinization and the range over which it occursis governed by starch concentration, method of observation, granuletype, and heterogeneities within the granule population underobservation”. A number of techniques are available for the determinationof gelatinization as induced by heating, a convenient and accuratemethod being differential scanning calorimetry, which detects thetemperature range and enthalpy associated with the collapse of molecularorders within the granule. To obtain accurate and meaningful results,the peak and/or onset temperature of the endotherm observed bydifferential scanning calorimetry is usually determined.

The consequence of the collapse of molecular orders within starchgranules is that the granules are capable of taking up water in aprocess known as pasting, which has been defined (W A Atwell et al,Cereal Foods World 33, 306-311, 1988) as “ . . . the phenomenonfollowing gelatinization in the dissolution of starch. It involvesgranular swelling, exudation of molecular components from the granule,and eventually, total disruption of the granules”. The best method ofevaluating pasting properties is considered to be the viscoamylograph(Atwell et al, 1988 cited above) in which the viscosity of a stirredstarch suspension is monitored under a defined time/temperature regime.A typical viscoamylograph profile for potato starch shows an initialrise in viscosity, which is considered to be due to granule swelling. Inaddition to the overall shape of the viscosity response in aviscoamylograph, a convenient quantitative measure is the temperature ofinitial viscosity development (onset). FIG. 1 shows such a typicalviscosity profile for potato starch, during and after cooking, andincludes stages A-D which correspond to viscosity onset (A), maximumviscosity (B), complete dispersion (C) and reassociation of molecules(or retrogradation, D). In the figure, the dotted line representsviscosity (in stirring number units) of a 10% w/w starch suspension andthe unbroken line shows the temperature in degrees centigrade. At acertain point, defined by the viscosity peak, granule swelling is soextensive that the resulting highly expanded structures are susceptibleto mechanically-induced fragmentation under the stirring conditionsused. With increased heating and holding at 95° C., further reduction inviscosity is observed due to increased fragmentation of swollengranules. This general profile has previously always been found fornative potato starch.

After heating starches in water to 95° C. and holding at thattemperature (for typically 15 minutes), subsequent cooling to 50° C.results in an increase in viscosity due to the process of retrogradationor set-back. Retrogradation (or set-back) is defined (Atwell et al.,1988 cited above) as “ . . . a process which occurs when the moleculescomprising gelatinised starch begin to reassociate in an orderedstructure . . . ”. At 50° C., it is primarily the amylose componentwhich reassociates, as indicated by the increase in viscoamylographviscosity for starch from normal maize (21.6% amylose) compared withstarch from waxy maize (1.1% amylose) as shown in FIG. 2. FIG. 2 is aviscoamylograph of 10% w/w starch suspensions from waxy maize (solidline), conventional maize (dots and dashes), high amylose variety(HYLON® V starch, dotted line) and a very high amylose variety (HYLON®VII starch, crosses). The temperature profile is also shown by a solidline, as in FIG. 1. The extent of viscosity increase in theviscoamylograph on cooling and holding at 50° C. depends on the amountof amylose which is able to reassociate due to its exudation from starchgranules during the gelatinization and pasting processes. Acharacteristic of amylose-rich starches from maize plants is that verylittle amylose is exuded from granules by gelatinization and pasting upto 95° C., probably due to the restricted swelling of the granules. Thisis illustrated in FIG. 2 which shows low viscosities for a high amylose(44.9%) starch (HYLON® V starch) from maize during gelatinization andpasting at 95° C. and little increase in viscosity on cooling andholding at 50° C. This effect is more extreme for a higher amylosecontent (58%, as in HYLON® VII starch), which shows even lowerviscosities in the viscoamylograph test (FIG. 2). Forcommercially-available high amylose starches (currently available frommaize plants, such as those described above), processing at greater than100° C. is usually necessary in order to generate the benefits of highamylose contents with respect to increased rates and strengths ofreassociation, but use of such high temperatures is energeticallyunfavourable and costly. Accordingly, there is an unmet need forstarches of high amylose content which can be processed below 100° C.and still show enhanced levels of reassociation, as indicated forexample by viscoamylograph measurements.

The properties of potato starch are useful in a variety of both food andnon-food (paper, textiles, adhesives etc.) applications. However, formany applications, properties are not optimum and various chemical andphysical modifications well known in the art are undertaken in order toimprove useful properties. Two types of property manipulation whichwould be of use are: the controlled alteration of gelatinization andpasting temperatures; and starches which suffer less granularfragmentation during pasting than conventional starches.

Currently the only ways of manipulating the gelatinization and pastingtemperatures of potato starch are by the inclusion of additives such assugars, polyhydroxy compounds of salts (Evans & Haisman, Starke 34,224-231, 1982) or by extensive physical or chemical pre-treatments (e.g.Stute, Starke 44, 205-214, 1992). The reduction of granule fragmentationduring pasting can be achieved either by extensive physicalpretreatments (Stute, Starke 44, 205-214, 1992) or by chemicalcross-linking. Such processes are inconvenient and inefficient. It istherefore desirable to obtain plants which produce starch whichintrinsically possesses such advantageous properties.

Starch consists of two main polysaccharides, amylose and amylopectin.Amylose is a generally linear polymer containing α-1,4 linked glucoseunits, while amylopectin is a highly branched polymer consisting of aα-1,4 linked glucan backbone with α-1,6 linked glucan branches. In mostplant storage reserves amylopectin constitutes about 75% of the starchcontent. Amylopectin is synthesized by the concerted action of solublestarch synthase and starch branching enzyme [α-1,4 glucan: α-1,4 glucan6-glycosyltransferase, EC 2.4.1.18]. Starch branching enzyme (SBE)hydrolyses α-1,4 linkages and rejoins the cleaved glucan, via an α-1,6linkage, to an acceptor chain to produce a branched structure. Thephysical properties of starch are strongly affected by the relativeabundance of amylose and amylopectin, and SBE is therefore a crucialenzyme in determining both the quantity and quality of starches producedin plant systems.

In most plants studied to date e.g. maize (Boyer & Preiss, 1978 Biochem.Biophys. Res. Comm. 80, 169-175), rice (Smyth, 1988 Plant Sci. 57, 1-8)and pea (Smith, Planta 175, 270-279), two forms of SBE have beenidentified, each encoded by a separate gene. A recent review by Burtonet al., (1995 The Plant Journal 7, 3-15) has demonstrated that the twoforms of SBE constitute distinct classes of the enzyme such that, ingeneral, enzymes of the same class from different plants may exhibitgreater similarity than enzymes of different classes from the sameplant. In their review, Burton et al. termed the two respective enzymefamilies class “A” and class “B”, and the reader is referred thereto(and to the references cited therein) for a detailed discussion of thedistinctions between the two classes. One general distinction of notewould appear to be the presence, in class A SBE molecules, of a flexibleN-terminal domain, which is not found in class B molecules. Thedistinctions noted by Burton et al. are relied on herein to define classA and class B SBE molecules, which terms are to be interpretedaccordingly.

However in potato, only one isoform of the SBE molecule (belonging toclass B) has thus far been reported and only one gene cloned (Blennow &Johansson, 1991 Phytochem. 30, 437-444, and Koβmann el al., 1991 Mol.Gen. Genet. 230, 39-44). Further, published attempts to modify theproperties of starch in potato plants (by preventing expression of thesingle known SBE) have generally not succeeded (e.g. Müller-Rober &Koβmann 1994 Plant Cell and Environment 17, 601-613).

SUMMARY OF THE INVENTION

In a first aspect the invention provides a nucleotide sequence encodingan effective portion of a class A starch branching enzyme (SBE)obtainable from potato plants.

Preferably the nucleotide sequence encodes a polypeptide comprising aneffective portion of the amino acid sequence shown in FIG. 5 (excludingthe sequence MNKRIDL, which does not represent part of the SBEmolecule), or a functional equivalent thereof (which term is discussedbelow). The amino acid sequence shown in FIG. 5 (Seq ID No. 15) includesa leader sequence which directs the polypeptide, when synthesised inpotato cells, to the amyloplast. Those skilled in the art will recognisethat the leader sequence is removed to produce a mature enzyme and thatthe leader sequence is therefore not essential for enzyme activity.Accordingly, an “effective portion” of the polypeptide is one whichpossesses sufficient SBE activity to complement the branching enzymemutation in E. coli KV 832 cells (described below) and which is activewhen expressed in E. coli in the phosphorylation stimulation assay. Anexample of an incomplete polypeptide which nevertheless constitutes an“effective portion” is the mature enzyme lacking the leader sequence. Byanalogy with the pea class A SBE sequence, the potato class A sequenceshown in FIG. 5 probably possesses a leader sequence of about 48 aminoacid residues, such that the N terminal amino acid sequence is thoughtto commence around the glutamic acid residue (E) at position 49 (EKSSYN. . . etc.). Those skilled in the art will appreciate that an effectiveportion of the enzyme may well omit other parts of the sequence shown inthe figure without substantial detrimental effect. For example, theC-terminal glutamic acid-rich region could be reduced in length, orpossibly deleted entirely, without abolishing class A SBE activity. Acomparison with other known SBE sequences, especially other class A SBEsequences (see for example, Burton et al, 1995 cited above), shouldindicate those portions which are highly conserved (and thus likely tobe essential for activity) and those portions which are less wellconserved (and thus are more likely to tolerate sequence changes withoutsubstantial loss of enzyme activity).

Conveniently the nucleotide sequence will comprise substantiallynucleotides 289 to 2790 of the DNA sequence (Seq ID No. 14) shown inFIG. 5 (which nucleotides encode the mature enzyme) or a functionalequivalent thereof, and may also include further nucleotides at the 5′or 3′ end. For example, for ease of expression, the sequence willdesirably also comprise an in-frame ATG start codon, and may also encodea leader sequence. Thus, in one embodiment, the sequence furthercomprises nucleotides 145 to 288 of the sequence shown in FIG. 5. Otherembodiments are nucleotides 228 to 2855 of the sequence labelled“psbe2con.seq” in FIG. 8, and nucleotides 57 to 2564 of the sequenceshown in FIG. 12 (preferably comprising an in-frame ATG start codon,such as the sequence of nucleotides 24 to 56 in the same Figure), orfunctional equivalents of the aforesaid sequences.

The term “functional equivalent” as applied herein to nucleotidesequences is intended to encompass those sequences which differ in theirnucleotide composition to that shown in FIG. 5 but which, by virtue ofthe degeneracy of the genetic code, encode polypeptides having identicalor substantially identical amino acid sequences. It is intended that theterm should also apply to sequences which are sufficiently homologous tothe sequence of the invention that they can hybridise to the complementthereof under stringent hybridisation conditions—such equivalents willpreferably possess at least 85%, more preferably at least 90%, and mostpreferably at least 95% sequence homology with the sequence of theinvention as exemplified by nucleotides 289 to 2790 of the DNA sequenceshown in FIG. 5. It will be apparent to those skilled in the art thatthe nucleotide sequence of the invention may also find usefulapplication when present as an “antisense” sequence. Accordingly,functionally equivalent sequences will also include those sequenceswhich can hybridise, under stringent hybridisation conditions, to thesequence of the invention (rather than the complement thereof). Such“antisense” equivalents will preferably possess at least 85%, morepreferably at least 90%, and most preferably 95% sequence homology withthe complement of the sequence of the invention as exemplified bynucleotides 289 to 2790 of the DNA sequence shown in FIG. 5. Particularfunctional equivalents are shown, for example, in FIGS. 8 and 10 (if onedisregards the various frameshift mutations noted therein).

The invention also provides vectors, particularly expression vectors,comprising the nucleotide sequence of the invention. The vector willtypically comprise a promoter and one or more regulatory signals of thetype well known to those skilled in the art. The invention also includesprovision of cells transformed (which term encompasses transduction andtransfection) with a vector comprising the nucleotide sequence of theinvention.

The invention further provides a class A SBE polypeptide, obtainablefrom potato plants. In particular the invention provides the polypeptidein substantially pure form, especially in a form free from otherplant-derived (especially potato plant-derived) components, which can bereadily accomplished by expression of the relevant nucleotide sequencein a suitable non-plant host (such as any one of the yeast strainsroutinely used for expression purposes, e.g. Pichia spp. orSaccharomyces spp). Typically the enzyme will substantially comprise thesequence of amino acid residues 49 to 882 shown in FIG. 5 (disregardingthe sequence MNKRIDL, which is not part of the enzyme), or a functionalequivalent thereof. The polypeptide of the invention may be used in amethod of modifying starch in vitro, comprising treating starch undersuitable conditions (e.g. appropriate temperature, pH, etc.) with aneffective amount of the polypeptide according to the invention.

The term “functional equivalent”, as applied herein to amino acidsequences, is intended to encompass amino acid sequences substantiallysimilar to that shown in FIG. 5, such that the polypeptide possessessufficient activity to complement the branching enzyme mutation in E.coli KV 832 cells (described below) and which is active in E. coli inthe phosphorylation stimulation assay. Typically such functionallyequivalent amino acid sequences will preferably possess at least 85%,more preferably at least 90%, and most preferably at least 95% sequenceidentity with the amino acid sequence of the mature enzyme (i.e. minusleader sequence) shown in FIG. 5. Those skilled in the art willappreciate that conservative substitutions may be made generallythroughout the molecule without substantially affecting the activity ofthe enzyme. Moreover, some non-conservative substitutions may betolerated, especially in the less highly conserved regions of themolecule. Such substitutions may be made, for example, to modifyslightly the activity of the enzyme. The polypeptide may, if desired,include a leader sequence, such as that exemplified by residues 1 to 48of the amino acid sequence shown in FIG. 5, although other leadersequences and signal peptides and the like are known and may beincluded.

A portion of the nucleotide sequence of the invention has beenintroduced into a plant and found to affect the characteristics of theplant. In particular, introduction of the sequence of the invention,operably linked in the antisense orientation to a suitable promoter, wasfound to reduce the amount of branched starch molecules in the plant.Additionally, it has recently been demonstrated in other experimentalsystems that “sense suppression” can also occur (i.e. expression of anintroduced sequence operably linked in the sense orientation caninterfere, by some unknown mechanism, with the expression of the nativegene), as described by Matzke & Matzke (1995 Plant Physiol. 107,679-685). Any one of the methods mentioned by Matzke & Matzke could, intheory, be used to affect the expression in a host of a homologous SBEgene.

It is believed that antisense methods are mainly operable by theproduction of antisense mRNA which hybridises to the sense mRNA,preventing its translation into functional polypeptide, possibly bycausing the hybrid RNA to be degraded (e.g. Sheehy et al., 1988 PNAS 85,8805-8809; Van der Krol et al., Mol. Gen. Genet. 220, 204-212). Sensesuppression also requires homology between the introduced sequence andthe target gene, but the exact mechanism is unclear. It is apparenthowever that, in relation to both antisense and sense suppression,neither a full length nucleotide sequence, nor a “native” sequence isessential. Preferably the “effective portion” used in the method willcomprise at least one third of the full length sequence, but by simpletrial and error other fragments (smaller or larger) may be found whichare functional in altering the characteristics of the plant.

Thus, in a further aspect the invention provides a method of alteringthe characteristics of a plant, comprising introducing into the plant aneffective portion of the sequence of the invention operably linked to asuitable promoter active in the plant. Conveniently the sequence will belinked in the anti-sense orientation to the promoter. Preferably theplant is a potato plant. Conveniently, the characteristic alteredrelates to the starch content and/or starch composition of the plant(i.e. amount and/or type of starch present in the plant). Preferably themethod of altering the characteristics of the plant will also comprisethe introduction of one or more further sequences, in addition to aneffective portion of the sequence of the invention. The introducedsequence of the invention and the one or more further sequences (whichmay be sense or antisense sequences) may be operably linked to a singlepromoter (which would ensure both sequences were transcribed atessentially the same time), or may be operably linked to separatepromoters (which may be necessary for optimal expression). Whereseparate promoters are employed they may be identical to each other ordifferent. Suitable promoters are well known to those skilled in the artand include both constitutive and inducible types. Examples include theCaMV 35S promoter (e.g. single or tandem repeat) and the patatinpromoter. Advantageously the promoter will be tissue-specific. Desirablythe promoter will cause expression of the operably linked sequence atsubstantial levels only in the tissue of the plant where starchsynthesis and/or starch storage mainly occurs. Thus, for example, wherethe sequence is introduced into a potato plant, the operably linkedpromoter may be tuber-specific, such as the patatin promoter.

Desirably, for example, the method will also comprise the introductionof an effective portion of a sequence encoding a class B SBE, operablylinked in the antisense orientation to a suitable promoter active in theplant. Desirably the further sequence will comprise an effective portionof the sequence encoding the potato class B SBE molecule. Convenientlythe further sequence will comprise an effective portion of the sequencedescribed by Blennow & Johansson (1991 Phytochem. 30, 437-444) or thatdisclosed in WO92/11375. More preferably, the further sequence willcomprise at least an effective portion of the sequence disclosed inInternational Patent Application No. WO 95/26407. Use of antisensesequences against both class A and class B SBE in combination has nowbeen found by the present inventors to result in the production ofstarch having very greatly altered properties (see below). Those skilledin the art will appreciate the possibility that, if the plant alreadycomprises a sense or antisense sequence which efficiently inhibits theclass B SBE activity, introduction of a sense or antisense sequence toinhibit class A SBE activity (thereby producing a plant with inhibitionof both class A and class B activity) might alter greatly the propertiesof the starch in the plant, without the need for introduction of one ormore further sequences. Thus the sequence of the invention isconveniently introduced into plants already having low levels of class Aand/or class B SBE activity, such that the inhibition resulting from theintroduction of the sequence of the invention is likely to have a morepronounced effect.

The sequence of the invention, and the one or more further sequences ifdesired, can be introduced into the plant by any one of a number ofwell-known techniques (e.g. Agrobacterium-mediated transformation, or by“biolistic” methods). The sequences are likely to be most effective ininhibiting SBE activity in potato plants, but theoretically could beintroduced into any plant. Desirable examples include pea, tomato,maize, wheat, rice, barley, sweet potato and cassava plants. Preferablythe plant will comprise a natural gene encoding an SBE molecule whichexhibits reasonable homology with the introduced nucleic acid sequenceof the invention.

In another aspect, the invention provides a plant cell, or a plant orthe progeny thereof, which has been altered by the method defined above.The progeny of the altered plant may be obtained, for example, byvegetative propagation, or by crossing the altered plant and reservingthe seed so obtained. The invention also provides parts of the alteredplant, such as storage organs. Conveniently, for example, the inventionprovides tubers comprising altered starch, said tubers being obtainedfrom an altered plant or the progeny thereof. Potato tubers obtainedfrom altered plants (or the progeny thereof) will be particularly usefulmaterials in certain industrial applications and for the preparationand/or processing of foodstuffs and may be used, for example, to preparelow-fat waffles and chips (amylose generally being used as a coating toprevent fat uptake), and to prepare mashed potato (especially “instant”mashed potato) having particular characteristics.

In particular relation to potato plants, the invention provides a potatoplant or part thereof which, in its wild type possesses an effective SBEA gene, but which plant has been altered such that there is no effectiveexpression of an SBE A polypeptide within the cells of at least part ofthe plant. The plant may have been altered by the method defined above,or may have been selected by conventional breeding to be deleted for theclass A SBE gene, presence or absence of which can be readily determinedby screening samples of the plants with a nucleic acid probe or antibodyspecific for the potato class A gene or gene product respectively.

The invention also provides starch extracted from a plant altered by themethod defined above, or the progeny of such a plant, the starch havingaltered properties compared to starch extracted from equivalent. butunaltered, plants. The invention further provides a method of makingaltered starch, comprising altering a plant by the method defined aboveand extracting therefrom starch having altered properties compared tostarch extracted from equivalent, but unaltered, plants. Use ofnucleotide sequences in accordance with the invention has allowed thepresent inventors to produce potato starches having a wide variety ofnovel properties.

In particular the invention provides the following: a plant (especiallya potato plant) altered by the method defined above, containing starchwhich, when extracted from the plant, has an elevated endotherm peaktemperature as judged by DSC, compared to starch extracted from asimilar, but unaltered, plant; a plant (especially a potato plant)altered by the method defined above, containing starch which, whenextracted from the plant, has an elevated viscosity onset temperature(conveniently elevated by 10-25° C.) as judged by viscoamylographcompared to starch extracted from a similar, but unaltered, plant; aplant (especially a potato plant) altered by the method defined above,containing starch which, when extracted from the plant. has a decreasedpeak viscosity (conveniently decreased by 240-700 SNUs) as judged byviscoamylograph compared to starch extracted from a similar, butunaltered, plant; a plant (especially a potato plant) altered by themethod defined above, containing starch which, when extracted from theplant, has an increased pasting viscosity (conveniently increased by37-260 SNUs) as judged by viscoamylograph compared to starch extractedfrom a similar, but unaltered, plant; a plant (especially a potatoplant) altered by the method defined above, containing starch which,when extracted from the plant, has an increased set-back viscosity(conveniently increased by 224-313 SNUs) as judged by viscoamylographcompared to starch extracted from a similar, but unaltered, plant; aplant (especially a potato plant) altered by the method defined above,containing starch which, when extracted from the plant, has a decreasedset-back viscosity as judged by viscoamylograph compared to starchextracted from a similar, but unaltered, plant; and a plant (especiallya potato plant) altered by the method defined above, containing starchwhich, when extracted from the plant, has an elevated amylose content asjudged by iodometric assay (i.e. by the method of Morrison & Laignelet1983, cited above) compared to starch extracted from a similar, butunaltered, plant. The invention also provides for starch obtainable orobtained from such plants as aforesaid.

In particular the invention provides for starch which, as extracted froma potato plant by wet milling at ambient temperature, has one or more ofthe following properties, as judged by viscoamylograph analysisperformed according to the conditions defined below: viscosity onsettemperature in the range 70-95° C. (preferably 75-95° C.); peakviscosity in the range 500-12 stirring number units; pasting viscosityin the range 214-434 stirring number units; set-back viscosity in therange 450-618 or 14-192 stirring number units; or displays nosignificant increase in viscosity during viscoamylograph. Peak, pastingand set-back viscosities are defined below. Viscosity onset temperatureis the temperature at which there is a sudden, marked increase inviscosity from baseline levels during viscoamylograph, and is a termwell-known to those skilled in the art.

In other particular embodiments, the invention provides starch which asextracted from a potato plant by wet milling at ambient temperature hasa peak viscosity in the range 200-500 SNUs and a set-back viscosity inthe range 275-618 SNUs as judged by viscoamylograph according to theprotocol defined below; and starch which as extracted from a potatoplant by wet milling at ambient temperature has a viscosity which doesnot decrease between the start of the heating phase (step 2) and thestart of the final holding phase (step 5) and has a set-back viscosityof 303 SNUs or less as judged by viscoamylograph according to theprotocol defined below.

For the purposes of the present invention, viscoamylograph conditionsare understood to pertain to analysis of a 10% (w/w) aqueous suspensionof starch at atmospheric pressure, using a Newport Scientific RapidVisco Analyser with a heating profile of: holding at 50° C. for 2minutes (step 1), heating from 50 to 95° C. at a rate of 1.5° C. perminute (step 2), holding at 95° C. for 15 minutes (step 3), cooling from95 to 50° C. at a rate of 1.5° C. per minute (step 4), and then holdingat 50° C. for 15 minutes (step 5). Peak viscosity may be defined forpresent purposes as the maximum viscosity attained during the heatingphase (step 2) or the holding phase (step 3) of the viscoamylograph.Pasting viscosity may be defined as the viscosity attained by the starchsuspensions at the end of the holding phase (step 3) of theviscoamylograph. Set-back viscosity may be defined as the viscosity ofthe starch suspension at the end of step 5 of the viscoamylograph.

In yet another aspect the invention provides starch from a potato planthaving an apparent amylose content (% w/w) of at least 35%, as judged byiodometric assay according to the method described by Morrison &Laignelet (1983 J. Cereal Science 1, 9-20). The iodometric assay isconducted by dissolving the starch in urea-dimethylsuphoxide (“UDMSO”),and aliquots of the solution are used to determine total amylose(measured on lipid-free starch, precipitated fromurea-dimethylsulphoxide solution with ethanol). UDMSO may be obtained bymixing 9 volumes of dimethylsulphoxide with 1 volume of 6-M urea.Aliquots of the starch-UDMSO solution are then treated with 1₂—KIreagent (2 mg I₂, 20 mg KI/ml) at different concentrations at constanttemperature and followed via colorimetry in order to determine the BlueValue. The Blue Value is defined as the absorbance/cm at 635 nm of 10 mganhydrous starch in 100 ml dilute I₂—KI solution at 20° C. Amylosecontent is calculated from the Blue Value according to the regressionequation: amylose (%)=(28.414×Blue Value)−6.218. Preferably the starchwill have an amylose content of at least 40%, more preferably at least50%, and most preferably at least 66%. Starch obtained directly from apotato plant and having such properties has not hitherto been produced.Indeed, as a result of the present invention, it is now possible togenerate in vivo potato starch which has some properties analogous tothe very high amylose starches (e.g. HYLON® VII starch) obtainable frommaize.

Starches with high (at least 35%) amylose contents find commercialapplication as, amongst other reasons, the amylose component of starchreassociates more strongly and rapidly than the amylopectin componentduring retrogradation processes. This may result, for example, in pasteswith higher viscosities, gels of greater cohesion, or films of greaterstrength for starches with high (at least 35%) compared with normal(less than 35%) amylose contents. Alternatively, starches may beobtained with very high amylose contents, such that the granulestructure is substantially preserved during heating, resulting in starchsuspensions which demonstrate substantially no increase in viscosityduring cooking (i.e. there is no significant viscosity increase duringviscoamylograph conditions defined above). Such starches typicallyexhibit a viscosity increase of less than 10% (preferably less than 5%)during viscoamylograph under the conditions defined above.

In commerce, these valuable properties are currently obtained fromstarches of high amylose content derived from maize plants. It would beof commercial value to have an alternative source of high amylosestarches from potato as other characteristics such as granule size,organoleptic properties and textural qualities may distinguishapplication performances of high amylose starches from maize and potatoplants.

Thus high amylose starch obtained by the method of the present inventionmay find application in many different technological fields, which maybe broadly categorised into two groups: food products and processing;and “Industrial” applications. Under the heading of food products, thenovel starches of the present invention may find application as, forexample, films, barriers, coatings or gelling agents. In general, highamylose content starches absorb less fat during frying than starcheswith low amylose content, thus the high amylose content starches of theinvention may be advantageously used in preparing low fat fried products(e.g. potato chips, crisps and the like). The novel starches may also beemployed with advantage in preparing confectionery and in granular andretrograded “resistant” starches. “Resistant” starch is starch which isresistant to digestion by α-amylase. As such, resistant starch is notdigested by α-amylases present in the human small intestine, but passesinto the colon where it exhibits properties similar to soluble andinsoluble dietary fibre. Resistant starch is thus of great benefit infoodstuffs due to its low calorific value and its high dietary fibrecontent. Resistant starch is formed by the retrogradation (akin torecrystallization) of amylose from starch gels. Such retrogradation isinhibited by amylopectin. Accordingly, the high amylose starches of thepresent invention are excellent starting materials for the preparationof resistant starch. Suitable methods for the preparation of resistantstarch are well-known to those skilled in the art and include, forexample, those described in U.S. Pat. Nos. 5,051,271 and 5,281,276.Conveniently the resistant starches provided by the present inventioncomprise at least 5% total dietary fibre, as judged by the method ofProsky et al., (1985 J. Assoc. Off. Anal. Chem. 68, 677), mentioned inU.S. Pat. No. 5,281, 276.

Under the heading of “Industrial” applications, the novel starches ofthe invention may be advantageously employed, for example, incorrugating adhesives, in biodegradable products such as loose fillpackaging and foamed shapes, and in the production of glass fibers andtextiles.

Those skilled in the art will appreciate that the novel starches of theinvention may, if desired, be subjected in vitro to conventionalenzymatic, physical and/or chemical modification, such as cross-linking,introduction of hydrophobic groups (e.g. octenyl succinic acid, dodecylsuccinic acid), or derivatization (e.g. by means of esterification oretherification).

In yet another aspect the invention provides high (35% or more) amylosestarches which generate paste viscosities greater than those obtainedfrom high amylose starches from maize plants after processing attemperatures below 100° C. This provides the advantage of moreeconomical starch gelatinization and pasting treatments through the useof lower processing temperatures than are currently required for highamylose starches from maize plants.

The invention will now be further described by way of illustrativeexample and with reference to the drawings, of which:

FIG. 1 shows a typical viscoamylograph for a 10% w/w suspension ofpotato starch;

FIG. 2 shows vsicoamylographs for 10% suspensions of starch from variousmaize varieties;

FIG. 3 is a schematic representation of the cloning strategy used by thepresent inventors;

FIG. 4a shows the amino acid alignment of the C-terminal portion ofstarch branching enzyme isoforms from various sources; amino acidresidues matching the consensus sequence are shaded;

FIG. 4b shows the alignment of DNA sequences of various starch branchingenzyme isoforms which encode a conserved amino acid sequence;

FIG. 5 shows the DNA sequence (Seq ID No. 14) and predicted amino acidsequence (Seq ID No. 15) of a full length potato class A SBE cDNA cloneobtained by PCR;

FIG. 6 shows a comparison of the most highly conserved part of the aminoacid sequences of potato class A (uppermost sequence) and class B(lowermost sequence) SBE molecules;

FIG. 7 shows a comparison of the amino acid sequence of the full lengthpotato class A (uppermost sequence) and pea (lowermost sequence) class ASBE molecules;

FIG. 8 shows a DNA alignment of various full length potato class A SBEclones obtained by the inventors;

FIG. 9 shows the DNA sequence of a potato class A SBE clone determinedby direct sequencing of PCR products, together with the predicted aminoacid sequence;

FIG. 10 is a multiple DNA alignment of various full length potato SBE Aclones obtained by the inventors;

FIG. 11 is a schematic illustration of the plasmid pSJ64;

FIG. 12 shows the DNA sequence and predicted amino acid sequence of thefull length potato class A SBE clone as present in the plasmid pSJ90;and

FIG. 13 shows vsicoamylographs for 10% w/w suspensions of starch fromvarious transgenic potato plants made by the relevant method aspect ofthe invention.

EXAMPLES

Prosky Method for Determining Dietary Fiber in Foods According toProsky, et al., J. Assoc. Off.

Anal. Chem., 68, 677 (1985)

Reagents:

(a) Ethanol 95% v/v, technical grade.

(b) Ethanol 78%. Place 207 ml water into a 1 L volume flask. Dilute tovolume with 95% EtOH. Mix and dilute to volume again with 95% EtOH ifnecessary. Mix

(c) Acetone, reagent grade.

(d) Phosphate buffer, 0.05M, pH 6.0. Dissolve 0.875 g Na phosphatedibasic, anhydride (Na₂HPO₄) (or 1.097 g dihydrate) and 6.05 g Naphosphate monobasic monohydrate (NaH₂PO₄) (or 6.84 g dihydrate) in a ca700 ml H.sub.2 O. Dilute to 1 L with H.sub.2 O. Check pH with pH meter.

(e) Termamyl (heat stable .alpha.-amylase) solution—No. 120 L, NovoLaboratories, Inc., Wilton Conn. 06897. Keep refrigerated.

(f) Protease. No. P-5380, Sigma Chemical Company. Keep refrigerated.

(g) Amyloglucosidase. No. A-9268, Sigma Chemical Company. Keeprefrigerated. Alternatively, a kit containing all 3 enzymes (pretested)is available from Sigma Chemical Company, Catalog No. KR-185.

(h) Sodium hydroxide solution, 0.171N. Dissolve 6.84 g NaOH ACS in ca700 ml water in 1 L. volume flask. Dilute to volume with water.

(i) Phosphoric acid solution, 0.205M. Dissolve 23.64 g H₃PO₄ ACS (85%)in water in 1 L volume flask. Dilute to volume with water.

(j) Celite C-211, acid-washed. Fisher Scientific Company.

Method

Run blank through entire procedure along with samples to measure anycontribution from reagents to residue. Homogenize sample and dryovernight in 70° C. vacuum oven, cool in desiccator, and dry-millportion of sample to 0.3-0.5 mm mesh. Weigh duplicate 1 g samples,accurate to 0.1 mg, into 400 ml, tall-form beakers. Sample weightsshould not differ by>20 mg. Add 50 ml pH 6.0 phosphate buffer to eachbeaker. Check pH and adjust if necessary. Add 0.1 ml Termanyl solution.Cover beaker with Aluminum foil and place in boiling water bath 15minutes. Shake gently at 5 minute intervals. Increase incubation timewhen number of beakers in boiling water bath makes it difficult forbeaker contents to reach internal temperature of 100° C. Use thermometerto ascertain that 100° is attained at 15 minutes. Total of 30 minutes inwater bath should be sufficient. Cool solutions to room temperature.Adjust to pH 7.5.+−.0.1 by adding 10 ml 0.171N NaOH solution. Add 5 mgprotease. (Protease sticks to spatula, so it may be preferable toprepare enzyme solution just before use with ca 0.1 ml phosphate bufferand pipet required amount).

Cover beaker with aluminum foil. Incubate 30 minutes at 60° C. withcontinuous agitation. Cool. Add 10 ml 0.205M H₃PO₄ solution to adjust pHto 4.5.+−.0.2. Add 0.3 ml amyloglucosidase, cover with aluminum foil andincubate 30 minutes at 60.degree. C. (Measure volume before heating.)Let precipitate form at room temperature for 60 minutes. Weigh cruciblecontaining Celite to nearest 0.1 mg, then wet and redistribute bed ofCelite in crucible by using stream of 78% EtOH from wash bottle. Applysuction to draw Celite onto fritted glass as even mat. Maintain suctionand quantitatively transfer precipitate from enzyme digest to crucible.Wash residue successively with three 20 ml portions of 78% EtOH, two 10ml portions of 95% EtOH, and two 10 ml portions of acetone. Gum may formwith some samples, trapping liquid. If so, break surface film withspatula to improve filtration. Time for filtration and washing will varyfrom 0.1-6 hours, averaging 1.2 hour per sample. Long filtration timescan be avoided by careful intermittent suction throughout filtration.

Dry crucible containing residue overnight in 70° C. vacuum oven or 105°C. air oven. Cool in desiccator and weigh to nearest 0.1 mg. Subtractcrucible and Celite weight to determine weight of residue. Analyzeresidue from sample of set of duplicates for protein and ash. Subtractprotein and ash values from residue to obtain TDF.

Determination of Blank

Blank=(mg blank residue−(% protein in blank+% ash in blank)×mg blankresidue)/100 Determination of TDF (%):

TDF%-mg residue−[((%protein in residue+% ash in residue)×mgresidue)−blank)×100/(mg sample (wt))]

EXAMPLE 1

Cloning of Potato Class A SBE

The strategy for cloning the second form of starch branching enzyme frompotato is shown in FIG. 3. The small arrowheads represent primers usedby the inventors in PCR and RACE protocols. The approximate size of thefragments isolated is indicated by the numerals on the right of theFigure. By way of explanation, a comparison of the amino acid sequencesof several cloned plant starch branching enzymes (SBE) from maize (classA), pea (class A), maize (class B), rice (class B) and potato (class B),as well as human glycogen branching enzyme, allowed the inventors toidentify a region in the carboxy-terminal one third of the protein whichis almost completely conserved (GYLNFMGNEFGHPEWIDFPR) (FIG. 4a). Amultiple alignment of the DNA sequences (human, pea class A, potatoclass B, maize class B, maize class A and rice class B, respectively)corresponding to this region is shown in FIG. 4b and was used to designan oligo which would potentially hybridize to all known plant starchbranching enzymes: AATTT(C/T)ATGGGIAA(C/T)GA(A/G)TT(C/T)GG (Seq ID No.20).

Library PCR

The initial isolation of a partial potato class A SBE cDNA clone wasfrom an amplified potato tuber cDNA library in the λZap vector(Stratagene). One half μL of a potato cDNA library (titre 2.3×10⁹pfu/mL)was used as template in a 50 μL reaction containing 100 pmol of a 16fold degenerate POTSBE primer and 25 pmol of a T7 primer (present in theλZap vector 3′ to the cDNA sequences—see FIG. 3), 100 μM dNTPs, 2.5 UTaq polymerase and the buffer supplied with the Taq polymerase(Stratagene). All components except the enzyme were added to a 0.5 mLmicrocentrifuge tube, covered with mineral oil and incubated at 94° C.for 7 minutes and then held at 55° C., while the Taq polymerase wasadded and mixed by pipetting. PCR was then performed by incubating for 1min at 94° C., 1 min at 58° C. and 3 minutes at 72° C., for 35 cycles.The PCR products were extracted with phenol/chloroform, ethanolprecipitated and resuspended in TE pH 8.0 before cloning into the T/Acloning vector pT7BlueR (Invitrogen).

Several fragments between 600 and 1300 bp were amplified. These wereisolated from an agarose gel and cloned into the pT7BlueR T/A cloningvector. Restriction mapping of 24 randomly selected clones showed thatthey belonged to several different groups (based on size andpresence/absence of restriction sites). Initially four clones werechosen for sequencing. Of these four, two were found to correspond tothe known potato class B SBE sequence, however the other two, althoughhomologous, differed significantly and were more similar to the peaclass A SBE sequence, suggesting that they belonged to the class Afamily of branching enzymes (Burton et al., 1995 The Plant Journal,cited above). The latter two clones (˜800bp) were sequenced fully. Theyboth contained at the 5′ end the sequence corresponding to thedegenerate oligonucleotide used in the PCR and had a predicted openreading frame of 192 amino acids. The deduced amino acid sequence washighly homologous to that of the pea class A SBE.

The ˜800 bp PCR derived cDNA fragment (corresponding to nucleotides 2281to 3076 of the psbe2 con.seq sequence shown in FIG. 8) was used as aprobe to screen the potato tuber cDNA library. From one hundred andeighty thousand plaques, seven positives were obtained in the primaryscreen. PCR analysis showed that five of these clones were smaller thanthe original 800 bp cDNA clone, so these were not analysed further. Thetwo other clones (designated 3.2.1 and 3.1.1) were approximately 1200and 1500 bp in length respectively. These were sequenced from their 5′ends and the combined consensus sequence aligned with the sequence fromthe PCR generated clones. The cDNA clone 3.2.1 was excised from thephage vector and plasmid DNA was prepared and the insert fullysequenced. Several attempts to obtain longer clones from the librarywere unsuccessful, therefore clones containing the 5′ end of the fulllength gene were obtained using RACE (rapid amplification of cDNA ends).

Rapid Amplification of cDNA Ends (RACE) and PCR Conditions

RACE was performed essentially according to Frohman (1992 Amplifications11-15). Two μg of total RNA from mature potato tubers was heated to 65°C. for 5 min and quick cooled on ice. The RNA was then reversetranscribed in a 20 μL reaction for 1 hour at 37° C. using BRL's M-MLVreverse transcriptase and buffer with 1 mM DTT, 1 mM dNTPs, 1 U/μLRNAsin (Promega) and 500 pmol random hexamers (Pharmacia) as primer.Excess primers were removed on a Centricon 100 column and cDNA wasrecovered and precipitated with isopropanol. cDNA was A-tailed in avolume of 20 μl using 10 units terminal transferase (BRL), 200 μM dATPfor 10 min at 37° C., followed by 5 min at 65° C. The reaction was thendiluted to 0.5 ml wi TE pH 8 and stored at 4° C. as the cDNA pool. cDNAclones were isolated by PCR amplification using the primers R_(o)R₁dT₁₇,R_(o) and POTSBE24. The PCR was performed in 50 μL using a hot starttechnique: 10 μL of the cDNA pool was heated to 94° C. in water for 5min with 25 pmol POTSBE24, 25 pmol R_(o) and 2.5 pmol of R_(o)R₁dT₁₇ andcooled to 75° C. Five μL of 10 ×PCR buffer (Stratagene), 200 μM dNTPsand 1.25 units of Taq polymerase were added, the mixture heated at 45°C. for 2 min and 72° C. for 40 min followed by 35 cycles of 94° C. for45 sec, 50° C. for 25 sec, 72° C. for 1.5 min and a final incubation at72° C. for 10 min. PCR products were separated by electrophoresis on 1%low melting agarose gels and the smear covering the range 600-800 bpfragments was excised and used in a second PCR amplification with 25pmol of R₁ and POTSBE25 primers in a 50 μL reaction (28 cycles of 94° C.for 1 min, 50° C. 1 min, 72° C. 2 min). Products were purified bychloroform extraction and cloned into pT7 Blue. PCR was used to screenthe colonies and the longest clones were sequenced.

The first round of RACE only extended the length of the SBE sequenceapproximately 100 bases, therefore a new A-tailed cDNA library wasconstructed using the class A SBE specific oligo POTSBE24 (10 pmol) inan attempt to recover longer RACE products. The first and second roundPCR reactions were performed using new class A SBE primers (POTSBE 28and 29 respectively) derived from the new sequence data. Conditions wereas before except that the elongation step in the first PCR was for 3 minand the second PCR consisted of 28 cycles at 94° C. for 45 seconds, 55°C. for 25 sec and 72° C. for 1 min 45 sec.

Clones ranging in size from 400 bp to 1.4 kb were isolated andsequenced. The combined sequence of the longest RACE products and cDNAclones predicted a full length gene of about 3150 nucleotides, excludingthe poly(A) tail (psbe 2con.seq in FIG. 8).

As the sequence of the 5′ half of the gene was compiled from thesequence of several RACE products generated using Taq polymerase, it waspossible that the compiled sequence did not represent that of a singlemRNA species and/or had nucleotide sequence changes. The 5′ 1600 basesof the gene was therefore re-isolated by PCR using Ultma, a thermostableDNA polymerase which, because it possesses a 3′-5′ exonuclease activity,has a lower error rate compared to Taq polymerase. Several PCR productswere cloned and restriction mapped and found to differ in the number ofHind III, Ssp I, and EcoR I sites. These differences do not representPCR artefacts as they were observed in clones obtained from independentPCR reactions (data not shown) and indicate that there are several formsof the class A SBE gene transcribed in potato tubers.

In order to ensure that the sequence of the full length cDNA clone wasderived from a single mRNA species it was therefore necessary to PCR theentire gene in one piece. cDNA was prepared according to the RACEprotocol except that the adaptor oligo R_(o)R₁dT₁₇ (5 pmol) was used asa primer and after synthesis the reaction was diluted to 200 μL with TEpH 8 and stored at 4° C. Two μL of the cDNA was used in a PCR reactionof 50 μL using 25 pmol of class A SBE specific primers PBER1 and PBERT(see below), and thirty cycles of 94° for 1 min, 60° C. for 1 min and72° C. for 3 min. If Taq polymerase was used the PCR products werecloned into pT7Blue whereas if Ultma polymerase was used the PCRproducts were purified by chloroform extraction, ethanol precipitationand kinased in a volume of 20 μL (and then cloned into pBSSK IIP whichhad been cut with EcoRV and dephosphorylated). At least four classes ofcDNA were isolated, which again differed in the presence or absence ofHind III, Ssp I and EcoR I sites. Three of these clones were sequencedfully, however one clone could not be isolated in sufficient quantity tosequence.

The sequence of one of the clones (number 19) is shown in FIG. 5. Thefirst methionine (initiation) codon starts a short open reading frame(ORF) of 7 amino acids which is out of frame with the next predicted ORFof 882 amino acids which has a molecular mass (Mr) of approximately 100Kd. Nucleotides 6-2996 correspond to SBE sequence—the rest of thesequence shown is vector derived. FIG. 6 shows a comparison of the mosthighly conserved part of the amino acid sequence of potato class A SBE(residues 180-871, top, row) and potato class B SBE (bottom row,residues 98-792); the middle row indicates the degree of similarity,identical residues being denoted by the common letter, conservativechanges by two dots and neutral changes by a single dot. Dashes indicategaps introduced to optimise the alignment. The class A SBE protein has44% identity over the entire length with potato class B SBE, and 56%identity therewith in the central conserved domain (FIG. 6), as judgedby the “Megalign” program (DNASTAR). However, FIG. 7 shows a comparisonbetwteen potato class A SBE (top row, residues 1-873) and pea class ASBE (bottom row, residues 1-861), from which it can be observed thatcloned potato gene is more homologous to the class A pea enzyme, wherethe identity is 70% over nearly the entire length, and this increases to83% over the central conserved region (starting at IPPP at position^(˜)170). It is clear from this analysis that this cloned potato SBEgene belongs to the class A family of SBE genes.

An E coli culture, containing the plasmid pSJ78 (which directs theexpression of a full length potato SBE Class A gene), has been deposited(on 3rd Jan. 1996) under the terms of the Budapest Treaty at TheNational Collections of Industrial and Marine Bacteria Limited (23 StMachar Drive, Aberdeen, AB2 1RY, United Kingdom), under accession numberNCIMB 40781. Plasmid pSJ78 is equivalent to clone 19 described above. Itrepresents a full length SBE A cDNA blunt-end ligated into the vectorpBSSKIIP.

Polymorphism of Class A SBE Genes

Sequence analysis of the other two full length class A SBE genes showedthat they contain frameshift mutations and are therefore unable toencode full length proteins and indeed they were unable to complementthe branching enzyme deficiency in the KV832 mutant (described below).An alignment of the full length DNA sequences is shown in FIG. 8:“10con.seq” (Seq ID No. 12), “19con.seq” (Seq ID No. 14) and “11con.seq”(Seq ID No. 13) represent the sequence of full length clones 10, 19 and11 obtained by PCR using the PBER1 and PBERT primers (see below), whilst“psbe2con.seq” (Seq ID No. 18) represents the consensus sequence of theRACE clones and cDNA clone 3.2.1. Those nucleotides which differ fromthe overall consensus sequence (not shown) are shaded. Dashes indicategaps introduced to optimise the alignment. Apart from the frameshiftmutations these clones are highly homologous. It should be noted thatthe 5′ sequence of psbe2con is longer because this is the longest RACEproduct and it also contains several changes compared to the otherclones. The upstream methionine codon is still present in this clone butthe upstream ORF is shortened to just 3 amino acids and in additionthere is a 10 base deletion in the 5′ untranslated leader.

The other significant area of variation is in the carboxy terminalregion of the protein coding region. Closer examination of this areareveals a GAA trinucleotide repeat structure which varies in lengthbetween the four clones. These are typical characteristics of amicrosatellite repeat region. The most divergent clone is #11 which hasonly one GAA triplet whereas clone 19 has eleven perfect repeats and theother two clones have five and seven GAA repeats. All of these deletionsmaintain the ORF but change the number of glutamic acid residues at thecarboxy terminus of the protein.

Most of the other differences between the clones are single basechanges. It is quite possible that some of these are PCR errors. Toaddress this question direct sequencing of PCR fragments amplified fromfirst strand cDNA was performed. FIG. 9 shows the DNA sequence, andpredicted amino acid sequence, obtained by such direct sequencing.Certain restriction sites are also marked. Nucleotides which could notbe unambiguously assigned are indicated using standard 1UPAC notationand, where this uncertainty affects the predicted amino acid sequence, aquestion mark is used. Sequence at the extreme 5′ and 3′ ends of thegene could not be determined because of the heterogeneity observed inthe different cloned genes in these regions (see previous paragraph).However this can be taken as direct evidence that these differences arereal and are not PCR or cloning artefacts.

There is absolutely no evidence for the frameshift mutations in the PCRderived sequence and it would appear that these mutations are anartefact of the cloning process, resulting from negative selectionpressure in E. coli. This is supported by the fact that it provedextremely difficult to clone the full length PCR products intact as manylarge deletions were seen and the full length clones obtained were allcloned in one orientation (away from the LacZ promoter), perhapssuggesting that expression of the gene is toxic to the cells.Difficulties of this nature may have been responsible, at least in part,for the previous failure of other researchers to obtain the presentinvention.

A comparison of all the full length sequences is shown in FIG. 10. Inaddition to clones 10, 11 and 19 are shown the sequences of a Bgl II—XhoI product cloned directly into the QE32 expression vector (“86CON.SEQ”,Seq ID No. 16) and the consensus sequence of the directly sequenced PCRproducts (“pcrsbe2con.seq”, Seq ID No. 17). Those nucleotides whichdiffer from the consensus sequence (not shown) are shaded. Dashesindicate gaps introduced to optimise the alignment. There are 11nucledtide differences predicted to be present in the mRNA population,which are indicated by asterisks above and below the sequence. The otherdifferences are probably PCR artefacts or possibly sequencing errors.

Complementation of a Branching Enzyme Deficient E. coli Mutant

To determine if the isolated SBE gene encodes an active protein i.e. onethat has branching enzyme activity, a complementation test was performedin the E. coli strain KV832. This strain is unable to make bacterialglycogen as the gene for the glycogen branching enzyme has been deleted(Keil et al., 1987 Mol. Gen. Genet. 207, 294-301). When wild type cellsare grown in the presence of glucose they synthesise glycogen (a highlybranched glucose polymer) which stains a brown colour with iodine,whereas the KV832 cells make only a linear chain glucose polymer whichstains bluish green with iodine. To determine if the cloned SBE genecould restore the ability of the KV832 cells to make a branched polymer,the clone pSJ90 (Seq ID No. 19) was used and constructed as below. Theconstruct is a PCR-derived, substantially full length fragment (madeusing primers PBE 2B and PBE 2X, detailed below), which was cut with BglII and Xho I and cloned into the BamH I/Sal I sites of the His-tagexpression vector pQE32 (Qiagen). This clone, pSJ86, was sequenced andfound to have a frameshift mutation of two bases in the 5′ half of thegene. This frameshift was removed by digestion with Nsi I and SnaB I andreplaced with the corresponding fragment from a Taq-generated PCR cloneto produce the plasmid pSJ90 (sequence shown in FIG. 12; the first 10amino acids are derived from the expression vector). The polypeptideencoded by pSJ90 would be predicted to correspond to amino acids 46-882of the full SBE coding sequence. The construct pSJ90 was transformedinto the branching enzyme deficient KV832 cells and transformants weregrown on solid PYG medium (0.85% KH₂PO₄, 1.1% K₂HPO₄, 0.6% yeastextract) containing 1.0% glucose. To test for complementation, a loop ofcells was scraped off and resuspended in 150 μl of water, to which wasadded 15 μl Lugol's solution (2 g KI and 1 g I₂ per 300 ml water). Itwas found that the potato SBE fragment-transformed KV832 cells nowstained a yellow-brown colour with iodine whereas control cellscontaining only the pQE32 vector continued to stain bluegreen.

Expression of Potato Class A SBE in E. coli

Single colonies of KV832, containing one of the plasmids pQE32, pAGCR1or pSJ90, were picked into 50 ml of 2×YT medium containingcarbenicillin, kanamycin and streptomycin as appropriate (100, 50 and 25mg/L, respectively) in a 250 ml flask and grown for 5 hours, withshaking, at 37° C. IPTG was then added to a final concentration of 1 mMto induce expression and the flasks were further incubated overnight at25° C. The cells were harvested by centrifugation and resuspended in 50mM sodium phosphate buffer (pH 8.0), containing 300 mM NaCl, 1 mg/mllysozyme and 1 mM PMSF and left on ice for 1 hour. The cell lysates werethen sonicated (3 pulses of 10 seconds at 40% power using a microprobe)and cleared by centrifugation at 12,000 g for 10 minutes at 4° C.Cleared lysates were concentrated approximately 10 fold in a Centricon™30 filtration unit. Duplicate 10 μl samples of the resulting extractwere assayed for SBE activity by the phosphorylation stimulation method,as described in International Patent Application No. PCT/GB95/00634. Inbrief, the standard assay reaction mixture (0.2 ml) was 200 mM2-(N-morpholino) ethanesulphonic acid (MES) buffer pH6.5, containing 100nCi of ¹⁴C glucose-1-phosphate at 50 mM, 0.05 mg rabbit phosphorylase A,and E. coli lysate. The reaction mixture was incubated for 60 minutes at30° C. and the reaction terminated and glucan polymer precipitated bythe addition of 1 ml of 75% (v/v) methanol, 1% (w/v) potassiumhydroxide, and then 0.1 ml glycogen (10 mg/ml). The results arepresented below:

Construct SBE Activity (cpm) pQE32 (control)  1,829 pSJ90 (potato classA SBE) 14,327 pAGCR1 (pea class A SBE) 29,707

The potato class A SBE activity is 7-8 fold above background levels. Itwas concluded therefore that the potato class A SBE gene was able tocomplement the BE mutation in the phosphorylation stimulation assay andthat the cloned gene does indeed code for a protein with branchingenzyme activity.

Oligonucleotides

The following synthetic oligonucleotides (Seq ID No.s 1-11 respectively)were used:

R_(O)R_(I)dT₁₇ AAGGATCCGTCGACATCGATAATACGACTCACTATAGGGA(T)₁₇ R_(O)AAGGATCCGTCGACATC R_(I) GACATCGATAATACGAC POTSBE24 CATCCAACCACCATCTCGCAPOTSBE25 TTGAGAGAAGATACCTAAGT POTSBE28 ATGTTCAGTCCATCTAAAGT POTSBE29AGAACAACAATTCCTAGCTC PBER 1 GGGGCCTTGAACTCAGCAAT PBERTCGTCCCAGCATTCGACATAA PBE 2B CTTGGATCCTTGAACTCAGCAATTTG PBE 2XTAACTCGAGCAACGCGATCACAAGTTCGT

Example 2

Production of Transgenic Plants

Construction of Plant Transformation Vectors with Antisense StarchBranching Enzyme Genes

A 1200 bp Sac I-Xho I fragment, encoding approximately the —COOH half ofthe potato class A SBE (isolated from the rescued λZap clone 3.2.1), wascloned into the Sac I-Sal I sites of the plant transformation vectorpSJ29 to create plasmid pSJ64, which is illustrated schematically inFIG. 11. In the figure, the black line represents the DNA sequence. Thebroken line represents the bacterial plasmid backbone (containing theorigin of replication and bacterial selection marker), which is notshown in full. The filled triangles on the line denote the T-DNA borders(RB=right border, LB=left border). Relevant restriction sites are shownabove the black line, with the approximate distances (in kilobases)between the sites (marked by an asterisk) given by the numerals belowthe line. The thinnest arrows indicate polyadenylation signals(pAnos=nopaline synthase, pAg7=Agrobacterium gene 7), the arrowsintermediate in thickness denote protein coding regions (SBE II=potatoclass A SBE, HYG=hygromycin resistance gene) and the thickest arrowsrepresent promoter regions (P-2×35 =double CaMV 35S promoter,Pnos=nopaline synthase promoter). Thus pSJ64 contained the class A SBEgene fragment in an antisense orientation between the 2X 35S CaMVpromoter and the nopaline synthase polyadenylation signal.

For information, pSJ29 is a derivative of the binary vector pGPTV-HYG(Becker et al., 1992 Plant Molecular Biology 20, 1195-1197) modified asfollows: an approximately 750 bp (Sac I, T4 DNA polymerase blunted—SalI) fragment of pJIT60 (Guerineau et al., 1992 Plant Mol. Biol. 18,815-818) containing the duplicated cauliflower mosaic virus (CaMV) 35Spromoter (Cabb-JI strain, equivalent to nucleotides 7040 to 7376duplicated upstream of 7040 to 7433, Frank et al., 1980 Cell 21,285-294) was cloned into the Hind III (Klenow polymerase repaired)—Sal Isites of pGPTV-HYG to create pSJ29.

Plant Transformation

Transformation was conducted on two types of potato plant explants;either wild type untransformed minitubers (in order to give singletransformants containing the class A antisense construct alone) orminitubers from three tissue culture lines (which gave rise to plants#12, #15, #17 and #18 indicated in Table 1) which had already beensuccessfully transformed with the class B (SBE I) antisense constructcontaining the tandem 35S promoter (so as to obtain double transformantplants, containing antisense sequences for both the class A and class Benzymes).

Details of the method of Agrobacterium transformation, and of the growthof transformed plants, are described in International Patent ApplicationNo. WO 95/26407, except that the medium used contained 3% sucrose (not1%) until the final transfer and that the initial incubation withAgrobacterium (strain 3850) was performed in darkness. Transformantscontaining the class A antisense sequence were selected by growth inmedium containing 15 mg/L hygromycin (the class A antisense constructcomprising the HYG gene, i.e. hygromycin phosphotransferase).

Transformation was confirmed in all cases by production of a DNAfragment from the antisense gene after PCR in the presence ofappropriate primers and a crude extract of genomic DNA from eachregenerated shoot.

Characterisation of Starch from Potato Plants

Starch was extracted from plants as follows: potato tubers werehomogenised in water for 2 minutes in a Waring blender operating at highspeed. The homogenate was washed and filtered (initially through 2 mm,then through 1 mm filters) using about 4 liters of water per 100 gms oftubers (6 extractions). Washed starch granules were finally extractedwith acetone and air dried.

Starch extracted from singly transformed potato plants (class A/SBE IIantisense, or class B/SBE I antisense), or from double transformants(class A/SBE II and class B/SBE I antisense), or from untransformedcontrol plants, was partially characterised. The results are shown inTable 1. The table shows the amount of SBE activity (units/gram tissue)in tubers from each transformed plant. The endotherm peak temperature (°C.) of starch extracted from several plants was determined by DSC, andthe onset temperature (° C) of pasting was determined by reference to aviscoamylograph (“RVA”), as described in WO 95/26407. Theviscoamylograph profile was as follows: step 1—50° C. for 2 minutes;step 2—increase in temperature from 50° C. to 95° C. at a rate of 1.5°C. per minute; step 3—holding at 95° C. for 15 minutes; step 4—coolingfrom 95° C. to 50° C. at a rate of 1.5° C. per minute; and finally, step5—holding at 50° C. for 15 minutes. Table 1 shows the peak, pasting andset-back viscosities in stirring number units (SNUs), which is a measureof the amount of torque required to stir the suspensions. Peak viscositymay be defined for present purposes as the maximum viscosity attainedduring the heating phase (step 2) or the holding phase (step 3) of theviscoamylograph. Pasting viscosity may be defined as the viscosityattained by the starch suspensions at the end of the holding phase (step3) of the viscoamylograph. Set-back viscosity may be defined as theviscosity of the starch suspension at the end of step 5 of theviscoamylograph.

A determination of apparent amylose content (% w/w) was also performed,using the iodometric assay method of Morrison & Laignelet (1983 J.Cereal Sci. 1, 9-20). The results (percentage apparent amylose) areshown in Table 1. The untransformed and transformed control plants gaverise to starches having apparent amylose contents in the range29(+/−3)%.

Generally similar values for amylose content were obtained for starchextracted from most of the singly transformed plants containing theclass A (SBE II ) antisense sequence. However, some plants (#152, 249)gave rise to starch having an apparent amylose content of 37-38%,notably higher than the control value. Starch extracted from theseplants had markedly elevated pasting onset temperatures, and starch fromplant 152 also exhibited an elevated endotherm peak temperature (starchfrom plant 249 was not tested by DSC).

TABLE 1 DSC Viscoamylograph (RVA) Peak Onset Apparent Tuber SBE temper-temper- Peak Pasting Set-back amylose Phosphorus Sample. activity atureature viscosity viscosity viscosity content content Sample descriptionnumber (U/g starch) (° C.) (° C.) (SNU) (SNU) (SNU) (% w/w) (mg/100 g)Untransformed control 146 7.6 65.8 65.5 545 161 260 31.2 68 243 22.2 nd62.6 761 135 241 29.1 AS-Class A SBE 152 12.7 69.5 70.9 467 380 529 37.589 249 13.9 nd 70.0 497 434 518 38.5 AS-Class B SBE (17) (control) 1450.7 66.9 66.8 669 177 305 29.8 111 AS-Class B SBE (17) + AS-Class A SBE150 0.6 74.0 86.0 214 214 303 53.1 198 161 0.5 73.0 76.6 349 324 61840.9 206 AS-Class B SBE (18) (control) 144 1.6 64.5 64.7 714 154 25829.0 97 AS-Class B SBE (18) + AS-Class A SBE 149 3.0 68.5 69.9 474 267482 35.6 127 AS-Class B SBE (15) (control) 172 0.22 nd 65.4 707 167 29028.8 130 AS-Class B SBE (15) + AS-Class A SBE 201 0.10 nd >95 no peak 1213 66.4 210 208a 0.10 nd >95 no peak 15 17 64.1 208 0.30 72.8-80.5 >95no peak 14 19 62.8 240 202 0.02 nd 89.4 no peak 172 245 57.9 212 1.40 nd78.0 308 296 541 49.5 220 1.40 nd 75.8 355 345 593 44.1 AS-Class B SBE(12) (control) 170 0.2 nd 66.5 768 202 303 27.8 AS-Class B SBE (12) +AS-Class A SBE 236 0.7 nd 95.0 no peak 23 14 60.4 236a 0.9 nd 91.2 nopeak 139 192 56.7 230a 0.8 nd 77.6 244 239 450 48.2 RVA profile 50° C.(2 min), 50-95° C. (1.5° C./min), 95° C. (15 min), 95-50° C. (1.5°C./min), 50° C. (15 min) Pasting viscosity (47 min) at end of 50° C. (2min), 50-95° C. (1.5° C./min), 95° C. (15 min) Set-back viscosity (92min) at end of profile SBE Starch Branching Enzyme SNU Instrument“Stirring Number Units” (arbitrary units) nd not determined

It should be noted that, even if other single transformants were not toprovide starch with an altered amylose/amylopectin ratio, the starchfrom such plants might still have different properties relative tostarch from conventional plants (e.g. different average molecular weightor different amylopectin branching patterns), which might be useful.

Double transformant plants, containing antisense sequences for both theclass A and class B enzymes, had greatly reduced SBE activity (units/gm)compared to untransformed plants or single anti-sense class Atransformants, (as shown in Table 1). Moreover, certain of the doubletransformant plants contained starch having very significantly alteredproperties. For example, starch extracted from plants #201, 202, 208,208a, 236 and 236a had drastically altered amylose/amylopectin ratios,to the extent that amylose was the main constituent of starch from theseplants. The pasting onset temperatures of starch from these plants werealso the most greatly increased (by about 25-30° C.). Starch from plantssuch as #150, 161, 212, 220 and 230a represented a range ofintermediates, in that such starch displayed a more modest rise in bothamylose content and pasting onset temperature. The results would tend tosuggest that there is generally a correlation between % amylose contentand pasting onset temperature, which is in agreement with the knownbehaviour of starches from other sources, notably maize.

The marked increase in amylose content obtained by inhibition of class ASBE alone, compared to inhibition of class B SBE alone (seePCT/GB95/00634) might suggest that it would be advantageous to transformplants first with a construct to suppress class A SBE expression(probably, in practice, an antisense construct), select those plantsgiving rise to starch with the most altered properties, and then tore-transform with a construct to suppress class B SBE expression (again,in practice, probably an antisense construct), so as to maximise thedegree of starch modification.

In addition to pasting onset temperatures, other features of theviscoamylograph profile e.g. for starches from plants #149, 150, 152,161, 201, 236 and 236a showed significant differences to starches fromcontrol plants, as illustrated in FIG. 13. Referring to FIG. 13, anumber of viscoamylograph traces are shown. The legend is as follows:shaded box—normal potato starch control (29.8% amylose content); shadedcircle—starch from plant 149 (35.6% amylose); shaded triangle, pointingupwards—plant 152 (37.5%); shaded triangle, pointing downwards—plant 161(40.9%); shaded diamond—plant 150 (53.1%); unshaded box—plant 236a(56.7%); unshaded circle—plant 236 (60.4%); unshaded triangle, pointingupwards—plant 201 (66.4%); unshaded triangle, pointing downwards—HYLON®V starch, from maize (44.9% amylose). The thin line denotes the heatingprofile.

With increasing amylose content, peak viscosities during processing to95° C. decrease, and the drop in viscosity from the peak until the endof the holding period at 95° C. also generally decreases (indeed, forsome of the starch samples there is an increase in viscosity during thisperiod). Both of these results are indicative of reduced granulefragmentation, and hence increased granule stability during pasting.This property has not previously been available in potato starch withoutextensive prior chemical or physical modification. For applicationswhere a maximal viscosity after processing to 95° C. is desirable (i.e.corresponding to the viscosity after 47 minutes in the viscoamylographtest), starch from plant #152 would be selected as starches with bothlower (Controls, #149) and higher (#161, #150) amylose contents havelower viscosities following this gelatinization and pasting regime (FIG.13 and Table 1). It is believed that the viscosity at this stage isdetermined by a combination of the extent of granule swelling and theresistance of swollen granules to mechanical fragmentation. For anydesired viscosity behaviour, one skilled in the art would select apotato starch from a range containing different amylose contentsproduced according to the invention by performing suitable standardviscosity tests.

Upon cooling pastes from 95° C. to 50° C., potato starches from mostplants transformed in accordance with the invention showed an increasein viscoamylograph viscosity as expected for partial reassociation ofamylose. Starches from plants #149, 152 and 161 all show viscosities at50° C. significantly in excess of those for starches from control plants(FIG. 13 and Table 1). This contrasts with the effect of elevatedamylose contents in starches from maize plants (FIG. 2) which show verylow viscosities throughout the viscoamylograph test. Of particular noteis the fact that, for similar amylose contents, starch from potato plant150 (53% amylose) shows markedly increased viscosity compared withHYLON® V starch (44.9% amylose) as illustrated in FIG. 13. Thisdemonstrates that useful properties which require elevated (35% orgreater) amylose levels can be obtained by processing starches frompotato plants below 100° C., whereas more energy-intensive processing isrequired in order to generate similarly useful properties from highamylose starches derived from maize plants.

Final viscosity in the viscoamylograph test (set-back viscosity after 92minutes) is greatest for starch from plant #161 (40.9% amylose) amongstthose tested (FIG. 13 and Table 1). Decreasing final viscosities areobtained for starches from plant #152 (37.5% amylose), #149 (35.6%amylose) and #150 (53.1% amy Set-back viscosity occurs where amylosemolecules, exuded from the starch granule during pasting, start tore-associate outside the granule and form a viscous gel-like substance.It is believed that the set-back viscosity values of starches fromtransgenic potato plants represent a balance between the inherentamylose content of the starches and the ability of the amylose fractionto be exuded from the granule during pasting and therefore be availablefor the reassociation process which results in viscosity increase. Forstarches with low amylose content, increasing the amylose content tendsto make more amylose available for re-association, thus increasing theset-back viscosity. However, above a threshold value, increased amylosecontent is thought to inhibit granule swelling, thus preventingexudation of amylose from the starch granule and reducing the amount ofamylose available for re-association. This is supported by the RVAresults obtained for the very high amylose content potato starches seenin the viscoamylograph profiles in FIG. 13. For any desired viscositybehaviour following set-back or retrogradation to any desiredtemperature over any desired timescale, one skilled in the art wouldselect a potato starch from a range containing different amylosecontents produced according to the invention by performing standardviscosity tests.

Further experiments with starch from plants #201 and 208 showed thatthis had an apparent amylose content of over 62% (see Table 1).Viscoamylograph studies showed that starch from these plants hadradically altered properties and behaved in a manner similar to HYLON® Vstarch from maize plants (FIG. 13). Under the conditions employed in theviscoamylograph, this starch exhibited extremely limited (nearlyundetectable) granule swelling. Thus, for example, unlike starch fromcontrol plants, starch from plants 201, 208 and 208a did not display aclearly defined pasting viscosity peak during the heating phase.Microscopic analysis confirmed that the starch granule structureunderwent only minor swelling during the experimental heating process.This property may well be particularly useful in certain applications,as will be apparent to those skilled in the art.

Some re-grown plants have so far been found to increase still furtherthe apparent amylose content of starch extracted therefrom. Suchincreases may be due to:-

i) Growth and development of the first generation transformed plants mayhave been affected to some degree by the exogenous growth hormonespresent in the tissue culture system, which exogenous hormones were notpresent during growth of the second generation plants; and

ii) Subsequent generations were grown under field conditions, which mayallow for attainment of greater maturity than growth under laboratoryconditions, it being generally held that amylose content of potatostarch increases with maturity of the potato tuber.

Accordingly, it should be possible to obtain potato plants giving riseto tubers with starch having an amylose content in excess of the 66%level so far attained, simply by analysing a greater number oftransformed plants and/or by re-growing transgenic plants through one ormore generations under field conditions.

Table 1 shows that another characteristic of starch which is affected bythe presence of anti-sense sequences to SBE is the phosphorus content.Starch from untransformed control plants had a phosphorus content ofabout 60-70 mg/100 gram dry weight (as determined according to the AOACOfficial Methods of Analysis, 15th Edition, Method 948.09 “Phosphorus inFlour”). Introduction into the plant of an anti-sense SBE B sequence wasfound to cause a modest increase (about two-fold) in phosphorus content,which is in agreement with the previous findings reported at scientificmeetings. Similarly, anti-sense to SBE A alone causes only a small risein phosphorus content relative to untransformed controls. However, useof anti-sense to both SBE A and B in combination results in up to afour-fold increase in phosphorus content, which is far greater than anyin planta phosphorus content previously demonstrated for potato starch.

This is useful in that, for certain applications, starch must bephosphorylated in vitro by chemical modification. The ability to obtainpotato starch which, as extracted from the plant, already has a highphosphorus content will reduce the amount of in vitro phosphorylationrequired suitably to modify the starch. Thus, in another aspect theinvention provides potato starch which, as extracted from the plant, hasa phosphorus content in excess of 200 mg/100 gram dry weight starch.Typically the starch will have a phosphorus content in the range 200-240mg/100 gram dry weight starch.

20 57 base pairs nucleic acid single linear 1 AAGGATCCGT CGACATCGATAATACGACTC ACTATAGGGA TTTTTTTTTT TTTTTTT 57 17 base pairs nucleic acidsingle linear 2 AAGGATCCGT CGACATC 17 17 base pairs nucleic acid singlelinear 3 GACATCGATA ATACGAC 17 20 base pairs nucleic acid single linear4 CATCCAACCA CCATCTCGCA 20 20 base pairs nucleic acid single linear 5TTGAGAGAAG ATACCTAAGT 20 20 base pairs nucleic acid single linear 6ATGTTCAGTC CATCTAAAGT 20 20 base pairs nucleic acid single linear 7AGAACAACAA TTCCTAGCTC 20 20 base pairs nucleic acid single linear 8GGGGCCTTGA ACTCAGCAAT 20 20 base pairs nucleic acid single linear 9CGTCCCAGCA TTCGACATAA 20 26 base pairs nucleic acid single linear 10CTTGGATCCT TGAACTCAGC AATTTG 26 29 base pairs nucleic acid single linear11 TAACTCGAGC AACGCGATCA CAAGTTCGT 29 3003 base pairs nucleic acidsingle linear 12 GATGGGGCCT TGAACTCAGC AATTTGACAC TCAGTTAGTT ACACTGCCATCACTTATCAG 60 ATCTCTATTT TTTCTCTTAA TTCCAACCAA GGAATGAATA AAAAGATAGATTTGTAAAAA 120 CCCTAAGGAG AGAAGAAGAA AGATGGTGTA TACACTCTCT GGAGTTCGTTTTCCTACTGT 180 TCCATCAGTG TACAAATCTA ATGGATTCAG CAGTAATGGT GATCGGAGGAATGCTAATAT 240 TTCTGTATTC TTGAAAAAAC ACTCTCTTTC ACGGAAGATC TTGGCTGAAAAGTCTTCTTA 300 CAATTCCGAA TCCCGACCTT CTACAATTGC AGCATCGGGG AAAGTCCTTGTGCCTGGAAT 360 CCAGAGTGAT AGCTCCTCAT CCTCAACAGA TCAATTTGAG TTCGCTGAGACATCTCCAGA 420 AAATTCCCCA GCATCAACTG ATGTAGATAG TTCAACAATG GAACACGCTAGCCAGATTAA 480 AACTGAGAAC GATGACGTTG AGCCGTCAAG TGATCTTACA GGAAGTGTTGAAGAGCTGGA 540 TTTTGCTTCA TCACTACAAC TACAAGAAGG TGGTAAACTG GAGGAGTCTAAAACATTAAA 600 TACTTCTGAA GAGACAATTA TTGATGAATC TGATAGGATC AGAGAGAGGGGCATCCCTCC 660 ACCTGGACTT GGTCAGAAGA TTTATGAAAT AGACCCCCTT TTGACAAACTATCGTCAACA 720 CCTTGATTAC AGGTATTCAC AGTACAAGAA ACTGAGGGAG GCAATTGACAAGTATGAGGG 780 TGGTTTGGAA GCTTTTTCTC GTGGTTATGA AAGAATGGGT TTCACTCGTAGTGCTACAGG 840 TATCACTTAC CGTGAGTGGG CTCCTGGTGC CCAGTCAGCT GCCCTCATTGGGGATTTCAA 900 CAATTGGGAC GCAAATGCTG ACTTTATGAC TCGGAATGAA TTTGGTGTCTGAGAGATTTT 960 TCTGCCAAAT AATGTGGATG GTTCTCCTGC AATTCCTCAT GGGTCCAGAGTGAAGATACG 1020 TATGGACACT CCATCAGGTG TTAAGGATTC CATTCCTGCT TGGATCAACTACTCTTTACA 1080 GCTTCCTGAT GAAATTCCAT ATAATGGAAT ATATTATGAT CCACCCGAAGAGGAGAGGTA 1140 TATCTTCCAA CACCCACGGC CAAAGAAACC AAAGTCGGTG AGAATATATGAATCTCATAT 1200 TGGAATGAGT AGTCCGGAGC CTAAAATTAA CTCATACGTG AATTTTAGAGATGAAGTTCT 1260 TCCTCGCATA AAAAAAGCTT GGGTACAATG CGGTGCAAAT TATGGCTATTCAAGAGCATT 1320 CTTATTATGC TAGTTTTGGT TATCATGTCA CAAATTTTTT TGCACCAAGCAGCCGTTTTG 1380 GAACGCCCGA CGACCTTAAG TCTTTGATTG ATAAAGCTCA TGAGCTAGGAATTGTTGTTC 1440 TCATGGACAT TGTTCACAGC CATGCATCAA ATAATACTTT AGATGGACTGAACATGTTTG 1500 ACGGCACAGA TAGTTGTTAC TTTCACTCTG GAGCTCGTGG TTATCATTGGATGTGGGATT 1560 TCCGCCTCTT TAACTATGGA AACTGGGAGG TACTTAGGTA TCTTCTCTCAAATGCGAGAT 1620 GGTGGTTGGA TGAGTTCAAA TTTGATGGAT TTAGATTTGA TGGTGTGACATCAATGATGT 1680 GTACTCACCA CGGATTATCG GTGGGATTCA CTGGGAACTA CGAGGAATACTTTGGACTCG 1740 CAACTGATGT GGATGCTGTT GTGTATCTGA TGCTGGTCAA CGATCTTATTCATGGGCTTT 1800 TCCCAGATGC AATTACCATT GGTGAAGATG TTAGCGGAAT GCCGACATTTTGTGTTCCCG 1860 TTCAAGATGG GGGTGTTGGC TTTGACTATC GGCTGCATAT GGCAATTGCTGATAAATGGA 1920 TTGAGTTGCT CAAGAAACGG GATGAGGATT GGAGAGTGGG TGATATTGTTCATACACTGA 1980 CAAATAGAAG ATGGTCGGAA AAGTGTGTTT CATACGCTGA AAGTCATGATCAAGCTCTAG 2040 TCGGTGATAA AACTATAGCA TTCTGGCTGA TGGACAAGGA TATGTATGATTTTATGGCTC 2100 TGGATAGACC GTCAACATCA TTAATAGATC GTGGGATAGC ATTACACAAGATGATTAGGC 2160 TTGTAACTAT GGGATTAGGA GGAGAAGGGT ACCTAAATTT CATGGGAAATGAATTCGGCC 2220 ACCCTGAGTG GATTGATTTC CCTAGGGCTG AACAACACCT CTCTGATGGCTCAGTAATTC 2280 CCAGAAACCA ATTCAGTTAT GATAAATGCA GACGGAGATT TGACCTGGGAGATGCAGAAT 2340 ATTTAAGATA CCGTGGGTTG CAAGAATTTG ACCGGGCTAT GCAGTATCTTGAAGATAAAT 2400 ATGAGTTTAT GACTTCAGAA CACCAGTTCA TATCACGAAA GGATGAAGGAGATAGGATGA 2460 TTGTATTTGA AAAAGGAAAC CTAGTTTTTG TCTTTAATTT TCACTGGACAAAAGGCTATT 2520 CAGACTATCG CATAGGCTGC CTGAAGCCTG GAAAATACAA GGTTGCCTTGGACTCAGATG 2580 ATCCACTTTT TGGTGGCTTC GGGAGAATTG ATCATAATGC CGAATATTTCACCTTTGAAG 2640 GATGGTATGA TGATCGTCCT CGTTCAATTA TGGTGTATGC ACCTAGTAGAACAGCAGTGG 2700 TCTATGCACT AGTAGACAAA GAAGAAGAAG AAGAAGAAGA AGTAGCAGTAGTAGAAGAAG 2760 TAGTAGTAGA AGAAGAATGA ACGAACTTGT GATCGCGTTG AAAGATTTGAACGCCACATA 2820 GAGCTTCTTG ACGTATCTGG CAATATTGCA TTAGTCTTGG CGGAATTTCATGTGACAACA 2880 GGTTTGCAAT TCTTTCCACT ATTAGTAGTG CAACGATATA CGCAGAGATGAAGTGCTGAA 2940 CAAAAACATA TGTAAAATCG ATGAATTTAT GTCGAATGCT GGGACGATCGAATTCCTGCA 3000 GCC 3003 2975 base pairs nucleic acid single linear 13TTGATGGGCC TTGAACTCAG CAATTTGACA CTCAGTTAGT TACACTCCTA TCACTTATCA 60GATCTCTATT TTTTCTCTTA ATTCCAACCA GGGGAATGAA TAAAAGGATA GATTTGTAAA 120AACCCTAAGG AGAGAAGAAG AAAGATGGTG TATATACTCT CTGGAGTTCG TTTTCCTACT 180GTTCCATCAG TGTACAAATC TAATGGATTC AGCAGTAATG GTGATCGGAG GAATGCTAAT 240GTTTCTGTAT TCTTGAAAAA GCACTCTCTT TCACGGAAGA TCTTGGCTGA AAAGTCTTCT 300TACAATTCCG AATTCCGACC TTCTACAGTT GCAGCATCGG GGAAAGTCCT TGTGCCTGGA 360ACCCAGAGTG ATAGCTCCTC ATCCTCAACA GACCAATTTG AGTTCACTGA GACATCTCCA 420GAAAATTCCC CAGCATCAAC TGATGTAGAT AGTTCAACAA TGGAACACGC TAGCCAGATT 480AAAACTGAGA ACGATGACGT TGAGCCGTCA AGTGATCTTA CAGGAAGTGT TGAAGAGCTG 540GATTTTGCTT CATCACTACA ACTACAAGAA GGTGGTAAAC TGGAGGAGTC TAAAACATTA 600AATACTTCTG AAGAGACAAT TATTGATGAA TCTGATAGGA TCAGAGAGAG GGGCATCCCT 660CCACCTGGAC TTGGTCAGAA GATTTATGAA ATAGACCCCC TTTTGACAAA CTATCGTCAA 720CACCTTGATT ACAGGTATTC ACAGTACAAG AAACTGAGGG AGGCAATTGA CAAGTATGAG 780GGTGGTTTGG AAGCTTTTCT CGTGGTTATG AAAAAATGGG TTTCACTCGT AGTGCTACAG 840GTATCACTTA CCGTGAGTGG GCTCCTGGTG CCCAGTCAGC TGCCCTCATT GGAGATTTCA 900ACAATTGGGA CGCAAATGCT GACATTATGA CTCGGAATGA ATTTGGTGTC TGGGAGATTT 960TTCTGCCAAA TAATGTGGAT GGTTCTCCTG CAATTCCTCA TGGGTCCAGA GTGAAGATAC 1020GTATGGACAC TCCATCAGGT GTTAAGGATT CCATTCCTGC TTGGATCAAC TACTCTTTAC 1080AGCTTCCTGA TGAAATTCCA TATAATGGAA TATATTATGA TCCACCCGAA GAGGAGAGGT 1140ATATCTTCCA ACACCCACGG CCAAAGAAAC CAAAGTCGCT GAGAATATAT GAATCTCATA 1200TTGGAATGAG TAGTCCGGAG CCTAAAATTA ACTCATACGT GAATTTTAGA GATGAAGTTC 1260TTCCTCGCAT AAAAAAGCTT GGGTACAATG CGCTGCGAAT TATGGCTATT CAAGAGCATT 1320CTTATTATGC TAGTTTTGGT TATCATGTCA CAAATTTTTT TGCACCAAGC AGCCGTTTTG 1380GAACGCCCGA CGACCTTAAG TCTTCGATTG ATAAAGCTCA TGAGCTAGGA ATTGTTGTTC 1440TCATGGACAT CGTTCACAGC CATGCATCAA ATAATACTTT AGATGGACTG AACATGTTTG 1500ACGGCACCGA TAGTTGTTAC TTTCACTCTG GAGCTCGTGG TTATCATTGG ATGTGGGATT 1560CCGCCTCTTT AACTATGGAA ACTGGGAGGT ACTTAGGTAT CTTCTCTCAA ATGCGAGATG 1620GTGGTTGGAT GAGTTCAAAT TTGATGGATT TAGATTCGAT GGTGTGACAT CAATGATGTA 1680TACTCACCAC GGATTATCGG TGGGATTCAC TGGGAACTAC GAGGAATACT TTGGACTCGC 1740AACTGATGTG GATGCTGTTG TGTATCTGAT GCTGGTCAAC GATCTTATTC ATAGGCTTTT 1800CCCAGATGCA ATTACCATTG GTGAAGATGT TAGCGGAATG CCGACATTTT GTATTCCCGT 1860TCAAGATGGG GGTGTTGGCT TTGACTATCG GCTGCATATG GCAATTGCTG ATAAATGGAT 1920TGAGTTGCTC AAGAAACGGG ATGAGGATTG GAGAGTGGGT GATATTGTTC ATACACTGAC 1980AAATAGAAGA TGGTCGGAAA AGTGTGTTTC ATACGCTGAA AGTCATGATC AAGCTCTAGT 2040CGGTGATAAA ACTATAGCAT TCTGGCTGAT GGACAAGGAT ATGTATGATT TTATGGCTCT 2100GGATAGACCG CCAACATCAT TAATAGATCG TGGGATAGCA TTGCACAAGA TGATTAGGCT 2160TGTAACTATG GGATTAGGAG GAGAAGGGTA CCTAAATTTC ATGGGAAATG AATTCGGCCA 2220CCCTGAGTGG ATTGATTTCC CTAGGGCTGA GCCACACCTT TCTGATGGCT CAGTAATTCC 2280CGGAAACCAA TTCAGTTATG ATAAATGCAG ACGGAGATTT GACCTGGGAG ATGCAGAATA 2340TTTAAGATAC CATGGGTTAC AAGAATTTGA CTGGGCTATG CAGTATCTTG AAGATAAATA 2400TGAGTTTATG ACTTCAGAAC ACCAGTTCAT ATCACGAAAG GATGAAGGAG ATAGGATGAT 2460TGTATTTGAA AGAGGAAACC TAGTTTTCGT CTTTAATTTT CACTGGACAA ATAGCTATTC 2520AGACTATCGC ATAGGCTGCC TGAAGCCTGG AAAATACAAG GTTGTCTTGG ACTCAGATGA 2580TCCACTTTTT GGTGGCTTCG GGAGAATTGA TCATAATGCC GAATATTTCA CCTCTGAAGG 2640ATCGTATGAT GATCGTCCTT GTTCAATTAT GGTGTATGCA CCTAGTAGAA CAGCAGTGGT 2700CTATGCACTA GTAGACAAAC TAGAAGTAGC AGTAGTAGAA GAACCCATTG AAGAATGAAC 2760GAACTTGTGA TCGCGTTGAA AGATTTGAAC GTTACTTGGT CATCCACATA GAGCTTCTTG 2820ACATCAGTCT TGGCGGAATT GCATGTGACA ACAAGGTTTG CAGTTCTTTC CACTATTAGT 2880AGTCCACCGA TATACGCAGA GATGAAGTGC TGAACAAACA TATGTAAAAT CGATGAATTT 2940ATGTCGAATG CTGGGACGAT CGAATTCCTG CAGCC 2975 3033 base pairs nucleic acidsingle linear CDS 145..2790 14 TTGATGGGGC CTTGAACTCA GCAATTTGACACTCAGTTAG TTACACTCCT ATCACTTATC 60 AGATCTCTAT TTTTTCTCTT AATTCCAACCAAGGAATGAA TAAAAGGATA GATTTGTAAA 120 AACCCTAAGG AGAGAAGAAG AAAG ATG GTGTAT ACA CTC TCT GGA GTT CGT 171 Met Val Tyr Thr Leu Ser Gly Val Arg 1 5TTT CCT ACT GTT CCA TCA GTG TAC AAA TCT AAT GGA TTC AGC AGT AAT 219 PhePro Thr Val Pro Ser Val Tyr Lys Ser Asn Gly Phe Ser Ser Asn 10 15 20 25GGT GAT CGG AGG AAT GCT AAT GTT TCT GTA TTC TTG AAA AAG CAC TCT 267 GlyAsp Arg Arg Asn Ala Asn Val Ser Val Phe Leu Lys Lys His Ser 30 35 40 CTTTCA CGG AAG ATC TTG GCT GAA AAG TCT TCT TAC AAT TCC GAA TTC 315 Leu SerArg Lys Ile Leu Ala Glu Lys Ser Ser Tyr Asn Ser Glu Phe 45 50 55 CGA CCTTCT ACA GTT GCA GCA TCG GGG AAA GTC CTT GTG CCT GGA ACC 363 Arg Pro SerThr Val Ala Ala Ser Gly Lys Val Leu Val Pro Gly Thr 60 65 70 CAG AGT GATAGC TCC TCA TCC TCA ACA GAC CAA TTT GAG TTC ACT GAG 411 Gln Ser Asp SerSer Ser Ser Ser Thr Asp Gln Phe Glu Phe Thr Glu 75 80 85 ACA TCT CCA GAAAAT TCC CCA GCA TCA ACT GAT GTA GAT AGT TCA ACA 459 Thr Ser Pro Glu AsnSer Pro Ala Ser Thr Asp Val Asp Ser Ser Thr 90 95 100 105 ATG GAA CACGCT AGC CAG ATT AAA ACT GAG AAC GAT GAC GTT GAG CCG 507 Met Glu His AlaSer Gln Ile Lys Thr Glu Asn Asp Asp Val Glu Pro 110 115 120 TCA AGT GATCTT ACA GGA AGT GTT GAA GAG CTG GAT TTT GCT TCA TCA 555 Ser Ser Asp LeuThr Gly Ser Val Glu Glu Leu Asp Phe Ala Ser Ser 125 130 135 CTA CAA CTACAA GAA GGT GGT AAA CTG GAG GAG TCT AAA ACA TTA AAT 603 Leu Gln Leu GlnGlu Gly Gly Lys Leu Glu Glu Ser Lys Thr Leu Asn 140 145 150 ACT TCT GAAGAG ACA ATT ATT GAT GAA TCT GAT AGG ATC AGA GAG AGG 651 Thr Ser Glu GluThr Ile Ile Asp Glu Ser Asp Arg Ile Arg Glu Arg 155 160 165 GGC ATC CCTCCA CCT GGA CTT GGT CAG AAG ATT TAT GAA ATA GAC CCC 699 Gly Ile Pro ProPro Gly Leu Gly Gln Lys Ile Tyr Glu Ile Asp Pro 170 175 180 185 CTT TTGACA AAC TAT CGT CAA CAC CTT GAT TAC AGG TAT TCA CAG TAC 747 Leu Leu ThrAsn Tyr Arg Gln His Leu Asp Tyr Arg Tyr Ser Gln Tyr 190 195 200 AAG AAACTG AGG GAG GCA ATT GAC AAG TAT GAG GGT GGT TTG GAA GCC 795 Lys Lys LeuArg Glu Ala Ile Asp Lys Tyr Glu Gly Gly Leu Glu Ala 205 210 215 TTT TCTCGT GGT TAT GAA AAA ATG GGT TTC ACT CGT AGT GCT ACA GGT 843 Phe Ser ArgGly Tyr Glu Lys Met Gly Phe Thr Arg Ser Ala Thr Gly 220 225 230 ATC ACTTAC CGT GAG TGG GCT CTT GGT GCC CAG TCA GCT GCC CTC ATT 891 Ile Thr TyrArg Glu Trp Ala Leu Gly Ala Gln Ser Ala Ala Leu Ile 235 240 245 GGA GATTTC AAC AAT TGG GAC GCA AAT GCT GAC ATT ATG ACT CGG AAT 939 Gly Asp PheAsn Asn Trp Asp Ala Asn Ala Asp Ile Met Thr Arg Asn 250 255 260 265 GAATTT GGT GTC TGG GAG ATT TTT CTG CCA AAT AAT GTG GAT GGT TCT 987 Glu PheGly Val Trp Glu Ile Phe Leu Pro Asn Asn Val Asp Gly Ser 270 275 280 CCTGCA ATT CCT CAT GGG TCC AGA GTG AAG ATA CGT ATG GAC ACT CCA 1035 Pro AlaIle Pro His Gly Ser Arg Val Lys Ile Arg Met Asp Thr Pro 285 290 295 TCAGGT GTT AAG GAT TCC ATT CCT GCT TGG ATC AAC TAC TCT TTA CAG 1083 Ser GlyVal Lys Asp Ser Ile Pro Ala Trp Ile Asn Tyr Ser Leu Gln 300 305 310 CTTCCT GAT GAA ATT CCA TAT AAT GGA ATA CAT TAT GAT CCA CCC GAA 1131 Leu ProAsp Glu Ile Pro Tyr Asn Gly Ile His Tyr Asp Pro Pro Glu 315 320 325 GAGGAG AGG TAT ATC TTC CAA CAC CCA CGG CCA AAG AAA CCA AAG TCG 1179 Glu GluArg Tyr Ile Phe Gln His Pro Arg Pro Lys Lys Pro Lys Ser 330 335 340 345CTG AGA ATA TAT GAA TCT CAT ATT GGA ATG AGT AGT CCG GAG CCT AAA 1227 LeuArg Ile Tyr Glu Ser His Ile Gly Met Ser Ser Pro Glu Pro Lys 350 355 360ATT AAC TCA TAC GTG AAT TTT AGA GAT GAA GTT CTT CCT CGC ATA AAA 1275 IleAsn Ser Tyr Val Asn Phe Arg Asp Glu Val Leu Pro Arg Ile Lys 365 370 375AAG CTT GGG TAC AAT GCG CTG CAA ATT ATG GCT ATT CAA GAG CAT TCT 1323 LysLeu Gly Tyr Asn Ala Leu Gln Ile Met Ala Ile Gln Glu His Ser 380 385 390TAT TAC GCT AGT TTT GGT TAT CAT GTC ACA AAT TTT TTT GCA CCA AGC 1371 TyrTyr Ala Ser Phe Gly Tyr His Val Thr Asn Phe Phe Ala Pro Ser 395 400 405AGC CGT TTT GGA ACG CCC GAC GAC CTT AAG TCT TTG ATT GAT AAA GCT 1419 SerArg Phe Gly Thr Pro Asp Asp Leu Lys Ser Leu Ile Asp Lys Ala 410 415 420425 CAT GAG CTA GGA ATT GTT GTT CTC ATG GAC ATT GTT CAC AGC CAT GCA 1467His Glu Leu Gly Ile Val Val Leu Met Asp Ile Val His Ser His Ala 430 435440 TCA AAT AAT ACT TTA GAT GGA CTG AAC ATG TTT GAC TGC ACC GAT AGT 1515Ser Asn Asn Thr Leu Asp Gly Leu Asn Met Phe Asp Cys Thr Asp Ser 445 450455 TGT TAC TTT CAC TCT GGA GCT CGT GGT TAT CAT TGG ATG TGG GAT TCC 1563Cys Tyr Phe His Ser Gly Ala Arg Gly Tyr His Trp Met Trp Asp Ser 460 465470 CGC CTC TTT AAC TAT GGA AAC TGG GAG GTA CTT AGG TAT CTT CTC TCA 1611Arg Leu Phe Asn Tyr Gly Asn Trp Glu Val Leu Arg Tyr Leu Leu Ser 475 480485 AAT GCG AGA TGG TGG TTG GAT GCG TTC AAA TTT GAT GGA TTT AGA TTT 1659Asn Ala Arg Trp Trp Leu Asp Ala Phe Lys Phe Asp Gly Phe Arg Phe 490 495500 505 GAT GGT GTG ACA TCA ATG ATG TAT ATT CAC CAC GGA TTA TCG GTG GGA1707 Asp Gly Val Thr Ser Met Met Tyr Ile His His Gly Leu Ser Val Gly 510515 520 TTC ACT GGG AAC TAC GAG GAA TAC TTT GGA CTC GCA ACT GAT GTG GAT1755 Phe Thr Gly Asn Tyr Glu Glu Tyr Phe Gly Leu Ala Thr Asp Val Asp 525530 535 GCT GTT GTG TAT CTG ATG CTG GTC AAC GAT CTT ATT CAT GGG CTT TTC1803 Ala Val Val Tyr Leu Met Leu Val Asn Asp Leu Ile His Gly Leu Phe 540545 550 CCA GAT GCA ATT ACC ATT GGT GAA GAT GTT AGC GGA ATG CCG ACA TTT1851 Pro Asp Ala Ile Thr Ile Gly Glu Asp Val Ser Gly Met Pro Thr Phe 555560 565 TGT ATT CCC GTC CAA GAG GGG GGT GTT GGC TTT GAC TAT CGG CTG CAT1899 Cys Ile Pro Val Gln Glu Gly Gly Val Gly Phe Asp Tyr Arg Leu His 570575 580 585 ATG GCA ATT GCT GAT AAA CGG ATT GAG TTG CTC AAG AAA CGG GATGAG 1947 Met Ala Ile Ala Asp Lys Arg Ile Glu Leu Leu Lys Lys Arg Asp Glu590 595 600 GAT TGG AGA GTG GGT GAT ATT GTT CAT ACA CTG ACA AAT AGA AGATGG 1995 Asp Trp Arg Val Gly Asp Ile Val His Thr Leu Thr Asn Arg Arg Trp605 610 615 TCG GAA AAG TGT GTT TCA TAC GCT GAA AGT CAT GAT CAA GCT CTAGTC 2043 Ser Glu Lys Cys Val Ser Tyr Ala Glu Ser His Asp Gln Ala Leu Val620 625 630 GGT GAT AAA ACT ATA GCA TTC TGG CTG ATG GAC AAG GAT ATG TATGAT 2091 Gly Asp Lys Thr Ile Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp635 640 645 TTT ATG GCT CTG GAT AGA CCG TCA ACA TCA TTA ATA GAT CGT GGGATA 2139 Phe Met Ala Leu Asp Arg Pro Ser Thr Ser Leu Ile Asp Arg Gly Ile650 655 660 665 GCA TTG CAC AAG ATG ATT AGG CTT GTA ACT ATG GGA TTA GGAGGA GAA 2187 Ala Leu His Lys Met Ile Arg Leu Val Thr Met Gly Leu Gly GlyGlu 670 675 680 GGG TAC CTA AAT TTC ATG GGA AAT GAA TTC GGC CAC CCT GAGTGG ATT 2235 Gly Tyr Leu Asn Phe Met Gly Asn Glu Phe Gly His Pro Glu TrpIle 685 690 695 GAT TTC CCT AGG GCT GAA CAA CAC CTC TCT GAT GGC TCA GTAATC CCC 2283 Asp Phe Pro Arg Ala Glu Gln His Leu Ser Asp Gly Ser Val IlePro 700 705 710 GGA AAC CAA TTC AGT TAT GAT AAA TGC AGA CGG AGA TTT GACCTG GGA 2331 Gly Asn Gln Phe Ser Tyr Asp Lys Cys Arg Arg Arg Phe Asp LeuGly 715 720 725 GAT GCA GAA TAT TTA AGA TAC CGT GGG TTG CAA GAA TTT GACCGG CCT 2379 Asp Ala Glu Tyr Leu Arg Tyr Arg Gly Leu Gln Glu Phe Asp ArgPro 730 735 740 745 ATG CAG TAT CTT GAA GAT AAA TAT GAG TTT ATG ACT TCAGAA CAC CAG 2427 Met Gln Tyr Leu Glu Asp Lys Tyr Glu Phe Met Thr Ser GluHis Gln 750 755 760 TTC ATA TCA CGA AAG GAT GAA GGA GAT AGG ATG ATT GTATTT GAA AAA 2475 Phe Ile Ser Arg Lys Asp Glu Gly Asp Arg Met Ile Val PheGlu Lys 765 770 775 GGA AAC CTA GTT TTT GTC TTT AAT TTT CAC TGG ACA AAAAGC TAT TCA 2523 Gly Asn Leu Val Phe Val Phe Asn Phe His Trp Thr Lys SerTyr Ser 780 785 790 GAC TAT CGC ATA GCC TGC CTG AAG CCT GGA AAA TAC AAGGTT GCC TTG 2571 Asp Tyr Arg Ile Ala Cys Leu Lys Pro Gly Lys Tyr Lys ValAla Leu 795 800 805 GAC TCA GAT GAT CCA CTT TTT GGT GGC TTC GGG AGA ATTGAT CAT AAT 2619 Asp Ser Asp Asp Pro Leu Phe Gly Gly Phe Gly Arg Ile AspHis Asn 810 815 820 825 GCC GAA TAT TTC ACC TTT GAA GGA TGG TAT GAT GATCGT CCT CGT TCA 2667 Ala Glu Tyr Phe Thr Phe Glu Gly Trp Tyr Asp Asp ArgPro Arg Ser 830 835 840 ATT ATG GTG TAT GCA CCT TGT AAA ACA GCA GTG GTCTAT GCA CTA GTA 2715 Ile Met Val Tyr Ala Pro Cys Lys Thr Ala Val Val TyrAla Leu Val 845 850 855 GAC AAA GAA GAA GAA GAA GAA GAA GAA GAA GAA GAAGAA GTA GCA GCA 2763 Asp Lys Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu GluVal Ala Ala 860 865 870 GTA GAA GAA GTA GTA GTA GAA GAA GAA TGAACGAACTTGTGATCGCG 2810 Val Glu Glu Val Val Val Glu Glu Glu 875 880 TTGAAAGATTTGAACGCTAC ATAGAGCTTC TTGACGTATC TGGCAATATT GCATCAGTCT 2870 TGGCGGAATTTCATGTGACA CAAGGTTTGC AATTCTTTCC ACTATTAGTA GTGCAACGAT 2930 ATACGCAGAGATGAAGTGCT GAACAAACAT ATGTAAAATC GATGAATTTA TGTCGAATGC 2990 TGGGACGATCGAATTCCTGC AGGCCGGGGG ACCCCTTAGT TCT 3033 882 amino acids amino acidlinear protein 15 Met Val Tyr Thr Leu Ser Gly Val Arg Phe Pro Thr ValPro Ser Val 1 5 10 15 Tyr Lys Ser Asn Gly Phe Ser Ser Asn Gly Asp ArgArg Asn Ala Asn 20 25 30 Val Ser Val Phe Leu Lys Lys His Ser Leu Ser ArgLys Ile Leu Ala 35 40 45 Glu Lys Ser Ser Tyr Asn Ser Glu Phe Arg Pro SerThr Val Ala Ala 50 55 60 Ser Gly Lys Val Leu Val Pro Gly Thr Gln Ser AspSer Ser Ser Ser 65 70 75 80 Ser Thr Asp Gln Phe Glu Phe Thr Glu Thr SerPro Glu Asn Ser Pro 85 90 95 Ala Ser Thr Asp Val Asp Ser Ser Thr Met GluHis Ala Ser Gln Ile 100 105 110 Lys Thr Glu Asn Asp Asp Val Glu Pro SerSer Asp Leu Thr Gly Ser 115 120 125 Val Glu Glu Leu Asp Phe Ala Ser SerLeu Gln Leu Gln Glu Gly Gly 130 135 140 Lys Leu Glu Glu Ser Lys Thr LeuAsn Thr Ser Glu Glu Thr Ile Ile 145 150 155 160 Asp Glu Ser Asp Arg IleArg Glu Arg Gly Ile Pro Pro Pro Gly Leu 165 170 175 Gly Gln Lys Ile TyrGlu Ile Asp Pro Leu Leu Thr Asn Tyr Arg Gln 180 185 190 His Leu Asp TyrArg Tyr Ser Gln Tyr Lys Lys Leu Arg Glu Ala Ile 195 200 205 Asp Lys TyrGlu Gly Gly Leu Glu Ala Phe Ser Arg Gly Tyr Glu Lys 210 215 220 Met GlyPhe Thr Arg Ser Ala Thr Gly Ile Thr Tyr Arg Glu Trp Ala 225 230 235 240Leu Gly Ala Gln Ser Ala Ala Leu Ile Gly Asp Phe Asn Asn Trp Asp 245 250255 Ala Asn Ala Asp Ile Met Thr Arg Asn Glu Phe Gly Val Trp Glu Ile 260265 270 Phe Leu Pro Asn Asn Val Asp Gly Ser Pro Ala Ile Pro His Gly Ser275 280 285 Arg Val Lys Ile Arg Met Asp Thr Pro Ser Gly Val Lys Asp SerIle 290 295 300 Pro Ala Trp Ile Asn Tyr Ser Leu Gln Leu Pro Asp Glu IlePro Tyr 305 310 315 320 Asn Gly Ile His Tyr Asp Pro Pro Glu Glu Glu ArgTyr Ile Phe Gln 325 330 335 His Pro Arg Pro Lys Lys Pro Lys Ser Leu ArgIle Tyr Glu Ser His 340 345 350 Ile Gly Met Ser Ser Pro Glu Pro Lys IleAsn Ser Tyr Val Asn Phe 355 360 365 Arg Asp Glu Val Leu Pro Arg Ile LysLys Leu Gly Tyr Asn Ala Leu 370 375 380 Gln Ile Met Ala Ile Gln Glu HisSer Tyr Tyr Ala Ser Phe Gly Tyr 385 390 395 400 His Val Thr Asn Phe PheAla Pro Ser Ser Arg Phe Gly Thr Pro Asp 405 410 415 Asp Leu Lys Ser LeuIle Asp Lys Ala His Glu Leu Gly Ile Val Val 420 425 430 Leu Met Asp IleVal His Ser His Ala Ser Asn Asn Thr Leu Asp Gly 435 440 445 Leu Asn MetPhe Asp Cys Thr Asp Ser Cys Tyr Phe His Ser Gly Ala 450 455 460 Arg GlyTyr His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Asn 465 470 475 480Trp Glu Val Leu Arg Tyr Leu Leu Ser Asn Ala Arg Trp Trp Leu Asp 485 490495 Ala Phe Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met 500505 510 Tyr Ile His His Gly Leu Ser Val Gly Phe Thr Gly Asn Tyr Glu Glu515 520 525 Tyr Phe Gly Leu Ala Thr Asp Val Asp Ala Val Val Tyr Leu MetLeu 530 535 540 Val Asn Asp Leu Ile His Gly Leu Phe Pro Asp Ala Ile ThrIle Gly 545 550 555 560 Glu Asp Val Ser Gly Met Pro Thr Phe Cys Ile ProVal Gln Glu Gly 565 570 575 Gly Val Gly Phe Asp Tyr Arg Leu His Met AlaIle Ala Asp Lys Arg 580 585 590 Ile Glu Leu Leu Lys Lys Arg Asp Glu AspTrp Arg Val Gly Asp Ile 595 600 605 Val His Thr Leu Thr Asn Arg Arg TrpSer Glu Lys Cys Val Ser Tyr 610 615 620 Ala Glu Ser His Asp Gln Ala LeuVal Gly Asp Lys Thr Ile Ala Phe 625 630 635 640 Trp Leu Met Asp Lys AspMet Tyr Asp Phe Met Ala Leu Asp Arg Pro 645 650 655 Ser Thr Ser Leu IleAsp Arg Gly Ile Ala Leu His Lys Met Ile Arg 660 665 670 Leu Val Thr MetGly Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly 675 680 685 Asn Glu PheGly His Pro Glu Trp Ile Asp Phe Pro Arg Ala Glu Gln 690 695 700 His LeuSer Asp Gly Ser Val Ile Pro Gly Asn Gln Phe Ser Tyr Asp 705 710 715 720Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala Glu Tyr Leu Arg Tyr 725 730735 Arg Gly Leu Gln Glu Phe Asp Arg Pro Met Gln Tyr Leu Glu Asp Lys 740745 750 Tyr Glu Phe Met Thr Ser Glu His Gln Phe Ile Ser Arg Lys Asp Glu755 760 765 Gly Asp Arg Met Ile Val Phe Glu Lys Gly Asn Leu Val Phe ValPhe 770 775 780 Asn Phe His Trp Thr Lys Ser Tyr Ser Asp Tyr Arg Ile AlaCys Leu 785 790 795 800 Lys Pro Gly Lys Tyr Lys Val Ala Leu Asp Ser AspAsp Pro Leu Phe 805 810 815 Gly Gly Phe Gly Arg Ile Asp His Asn Ala GluTyr Phe Thr Phe Glu 820 825 830 Gly Trp Tyr Asp Asp Arg Pro Arg Ser IleMet Val Tyr Ala Pro Cys 835 840 845 Lys Thr Ala Val Val Tyr Ala Leu ValAsp Lys Glu Glu Glu Glu Glu 850 855 860 Glu Glu Glu Glu Glu Glu Val AlaAla Val Glu Glu Val Val Val Glu 865 870 875 880 Glu Glu 2576 base pairsnucleic acid single linear 16 TCATTAAAGA GGAGAAATTA ACTATGAGAGGATCTCACCA TCACCATCAC CATGGGATCT 60 TGGCTGAAAA GTCTTCTTAC AATTCCGAATTCCGACCTTC TACAGTTGCA GCATCGGGGA 120 AAGTCCTTGT GCCTGGAACC CAGAGTGATAGCTCCTCATC CTCAACAAAC CAATTTGAGT 180 TCACTGAGAC ATCTCCAGAA AATTCCCCAGCATCAACTGA TGTAGATAGT TCAACAATGG 240 AACACGCTAG CCAGATTAAA ACTGAGAACGATGACGTTGA GCCGTCAAGT GATCTTACAG 300 GAAGTGTTGA AGAGCTGGAT TTTGCTTCATCACTACAACT ACAAGAAGGT GGTAAACTGG 360 AGGAGTCTAA AACATTAAAT ACTTCTGAAGAGACAATTAT TGATGAATCT GATAGGATCA 420 GAGAGAGGGG CATCCCTCCA CCTGGACTTGGTCAGAAGAT TTATGAAATA GACCCCCTTT 480 TGACAAACTA TCGTCAACAC CTTGATTACAGGTATTCACA GTACAAGAAA CTGAGGGAGG 540 CAATTGACAA GTATGAGGGT GGTTTGGAAGCTTTTTCTCG TGGTTATGAA AAAATGGGTT 600 TCACTCGTAG TGCTACAGGT ATCACTTACCGTGAGTGGGC TCCTGGTGCC CAGTCAGCTG 660 CCCTCATTGG AGATTTCAAC AATTGGGACGCAAATGCTGA CATTATGACT CGGAATGAAT 720 TTGGTGTCTG GGAGATTTTT CTGCCAAATAATGTGGATGG TTCTCCTGCA ATTCCTCATG 780 GGTCCAGAGT GAAGATACGT ATGGACACTCCATCAGGTGT TAAGGATTCC ATTCCTGCTT 840 GGATCAACTA CTCTACAGCT TCCTGATGAAATTCCATATA ATGGAATATA TTATGATCCA 900 CCCGAAGAGG AGAGGTATAT CTTCCAACACCCACGGCCAA AGAAACCAAA GTCGCTGAGA 960 ATATATGAAT CTCATATTGG AATGAGTAGTCCGGAGCCTA AAATTAACTC ATACGTGAAT 1020 TTTAGAGATG AAGTTCTTCC TCGCATAAAAAAGCTTGGGT ACAATGCGCT GCAAATTATG 1080 GCTATTCAAG AGCATTCTTA TTATGCTAGTTTTGGTTATC ATGTCACAAA TTTTTTTGCA 1140 CCAAGCAGCC GTTTTGGAAC GCCCGACGACCTTAAGTCTT TGATTGATAA AGCTCATGAG 1200 CTAGGAATTG TTGTTCTCAT GGACATTGTTCACAGCCATG CATCAAATAA TACTTTAGAT 1260 GGACTGAACA TGTTTGACGG CACCGATAGTTGTTACTTTC ACTCTGGAGC TCGTGGTTAT 1320 CATTGGATGT GGGATTCCCG CCTTTTTAACTATGGAAACT GGGAGGTACT TAGGTATCTT 1380 CTCTCAAATG CGAGATGGTG GTTGGATGAGTTCAAATTTG ATGGATTTAG ATTTGATGGT 1440 GTGACATCAA TGATGTATAC TCACCACGGATTATCGGTGG GATTCACTGG GAACTACGAG 1500 GAATACTTTG GACTCGCAAC TGATGTGGATGCTGTTGTGT ATCTGATGCT GGTCAACGAT 1560 CTTATTCATG GGCTTTTCCC AGATGCAATTACCATTGGTG AAGATGTTAG CGGAATGCCG 1620 ACATTTTGTA TTCCCGTTCA AGATGGGGGTGTTGGCTTTG ACTATCGGCT GCATATGGCA 1680 ATTGCTGATA AATGGATTGA GTTGCTCAAGAAACGGGATG AGGATTGGAG AGTGGGTGAT 1740 ATTGTTCATA CACTGACAAA TAGAAGATGGTCGGAAAAGT GTGTTTCATA CGCTGAAAGT 1800 CATGATCAAG CTCTAGTCGG TGATAAAACTATAGCATTCT GGCTGATGGA CAAGGATATG 1860 TATGATTTTA TGGCTCTGGA TAGACCGCCAACATCATTAA TAGATCGTGG GATAGCATTG 1920 CACAAGATGA TTAGGCTTGT AACTATGGGATTAGGAGGAG AAGGGTACCT AAATTTCATG 1980 GGAAATGAAT TCGGCCACCC TGAGTGGATTGATTTCCCTA GGGCTGAACA ACACCTCTCT 2040 GATGACTCAG TAATTCCCGG AAACCAATTCAGTTATGATA AATGCAGACG GAGATTTGAC 2100 CTGGGAGATG CAGAATATTT AAGATACCGTGGGTTGCAAG AATTTGACCG GGCTATGCAG 2160 TATCTTGAAG ATAAATATGA GTTTATGACTTCAGAACACC AGTTCATATC ACGAAAGGAT 2220 GAAGGAGATA GGATGATTGT ATTTGAAAAAGGAAACCTAG TTTTTGTCTT TAATTTTCAC 2280 TGGACAAAAA GCTATTCAGA CTATCGCATAGGCTGCCTGA AGCCTGGAAA ATACAAGGTT 2340 GCCTTGGACT CAGATGATCC ACTTTTTGGTGGCTTCGGGA GAATTGATCA TAATGCCGAA 2400 TATTTCACCT TTGAAGGATG GTATGATGATCGTCCTCGTT CAATTATGGT GTATGCACCT 2460 TGTAGAACAG CAGTGGTCTA TGCACTAGTAGACAAAGAAG AAGAAGAAGA AGAAGAAGAA 2520 GAAGAAGTAG CAGTAGTAGA AGAAGTAGTAGTAGAAGAAG AATGAACGAA CTTGTG 2576 2529 base pairs nucleic acid singlelinear 17 GGATGCTAAT GTTTCTGTAT TCTTGAAAAA GCACTCTCTT TCACGGAAGATCTTGGCTGA 60 AAAGTCTTCT TACAATTCCG AATCCCGACC TTCTACAGTT GCAGCATCGGGGAAAGTCCT 120 TGTGCCTGGA AYCCAGAGTG ATAGCTCCTC ATCCTCAACA GACCAATTTGAGTTCACTGA 180 GACATCTCCA GAAAATTCCC CAGCATCAAC TGATGTAGAT AGTTCAACAATGGAACACGC 240 TAGCCAGATT AAAACTGAGA ACGATGACGT TGAGCCGTCA AGTGATCTTACAGGAAGTGT 300 TGAAGAGCTG GATTTTGCTT CATCACTACA ACTACAAGAA GGTGGTAAACTGGAGGAGTC 360 TAAAACATTA AATACTTCTG AAGAGACAAT TATTGATGAA TCTGATAGGATCAGAGAGAG 420 GGGCATCCCT CCACCTGGAC TTGGTCAGAA GATTTATGAA ATAGACCCCCTTTTGACAAA 480 CTATCGTCAA CACCTTGATT ACAGGTATTC ACAGTACAAG AAACTGAGGGAGGCAATTGA 540 CAAGTATGAG GGTGGTTTGG AAGCTTTTTC TCGTGGTTAT GAAAAAATGGGTTTCACTCG 600 TAGTGCTACA GGTATCACTT ACCGTGAGTG GGCTCCTGGT GCCCAGTCAGCTGCCCTCAT 660 TGGAGATTTC AACAATTGGG ACGCAAATGC TGACATTATG ACTCGGAATGAATTTGGTGT 720 CTGGGAGATT TTTCTGCCAA ATAATGTGGA TGGTTCTCCT GCAATTCCTCATGGGTCCAG 780 AGTGAAGATA CGYATGGACA CTCCATCAGG TGTTAAGGAT TCCATTCCTGCTTGGATCAA 840 CTACTCTTTA CAGCTTCCTG ATGAAATTCC ATATAATGGA ATATATTATGATCCACCCGA 900 AGAGGAGAGG TATRTCTTCC AACACCCACG GCCAAAGAAA CCAAAGTCGCTGAGAATATA 960 TGAATCTCAT ATTGGAATGA GTAGTCCGGA GCCTAAAATT AACTCATACGTGAATTTTAG 1020 AGATGAAGTT CTTCCTCGCA TAAAAAASCT TGGGTACAAT GCGGTGCAAATTATGGCTAT 1080 TCAAGAGCAT TCTTATTATG CTAGTTTTGG TTATCATGTC ACAAATTTTTTTGCACCAAG 1140 CAGCCGTTTT GGAACGCCCG ACGACCTTAA GTCTTTGATT GATAAAGCTCATGAGCTAGG 1200 AATTGTTGTT CTCATGGACA TTGTTCACAG CCATGCATCA AATAATACTTTAGATGGACT 1260 GAACATGTTT GACGGCACAG ATAGTTGTTA CTTTCACTCT GGAGCTCGTGGTTATCATTG 1320 GATGTGGGAT TCCCGCCTCT TTAACTATGG AAACTGGGAG GTACTTAGGTATCTTCTCTC 1380 AAATGCGAGA TGGTGGTTGG ATGAGTTCAA ATTTGATGGA TTTAGATTTGATGGTGTGAC 1440 ATCAATGATG TATACTCACC ACGGATTATC GGTGGGATTC ACTGGGAACTACGAGGAATA 1500 CTTTGGACTC GCAACTGATG TGGATGCTGT TGTGTATCTG ATGCTGGTCAACGATCTTAT 1560 TCACGGGCTT TTCCCAGATG CAATTACCAT TGGTGAAGAT GTTAGCGGAATGCCGACATT 1620 TTGTATTCCC GTTCAAGATG GGGGTGTTGG CTTTGACTAT CGGCTGCATATGGCAATTGC 1680 TGATAAATGG ATTGAGTTGC TCAAGAAACG GGATGAGGAT TGGAGAGTGGGTGATATTGT 1740 TCATACACTG ACAAATAGAA GATGGTCGGA AAAGTGTGTT TCATMCGCTGAAAGTCATGA 1800 TCAAGCTCTA GTCGGTGATA AAACTATAGC ATYCTGGCTG ATGGACAAGGATATGTATGA 1860 TTTTATGGCT CTGGATAGAC CGYCAACAYC ATTAATAGAT CGTGGGATAGCATTGCACAA 1920 GATGATTAGG CTTGTAACTA TGGGATTAGG AGGAGAAGGG TACCTAAATTTCATGGGAAA 1980 TGAATTCGGC CACCCTGAGT GGATTGATTT CCCTAGGGCT GARCAACACCTCTCTGATGG 2040 CTCAGTAATT CCCGGAAACC AATTCAGTTA TGATAAATGC AGACGGAGATTTGACCTGGG 2100 AGATGCAGAA TATTTAAGAT ACCATGGGTT GCAAGAATTT GACCGGGCTATGCAGTATCT 2160 TGAAGATAAA TATGAGTTTA TGACTTCAGA ACACCAGTTC ATATCACGAAAGGATGAAGG 2220 AGATAGGATG ATTGTATTTG AAARAGGAAA CCTAGTTTTT GTCTTTAATTTTCACTGGAC 2280 AAATAGCTAT TCAGACTATC GCATAGGCTG CCTGAAGCCT GGAAAATACAAGGTTGGCTT 2340 GGACTCAGAT GATCCACTTT TTGGTGGCTT CGGGAGAATT GATCATAATGCCGAATATTT 2400 CACCTCTGAA GGATCGTATG ATGATCGTCC TCGTTCAATT ATGGTGTATGCACCTAGTAG 2460 AACAGCAGTG GTCTATGCAC TAGTAGACAA ANTAGAAGNA GAAGAAGAAGAAGAANCCGN 2520 NGAAGAATT 2529 3231 base pairs nucleic acid singlelinear 18 GATTTAATAC GACTCACTAT AGGGATTTTT TTTTTTTTTT TTTTAAAAACCTCCTCCACT 60 CAGTCTTGGG ATCTCTCTCT CTCTTCACGC TTCTCTTGGG GCCTTGAACTCAGCAATTTG 120 ACACTCAGTT AGTTACACTC CTATCACTCA TCAGATCTCT ATTTTTTCTCTTAATTCCAA 180 CCAAGGAATG AATTAAAAGA TTAGATTTGA AGGAGAGAAG AAGAAAGATGGTGTATACAC 240 TCTCTGGAGT TCGTTTTCCT ACTGTTCCAT CAGTGTACAA ATCTAATGGATTCAGCAGTA 300 ATGGTGATCG GAGGAATGCT AATGTTTCTG TATTCTTGAA AAAGCACTCTCTTTCACGGA 360 AGATCTTGGC TGAAAAGTCT TCTTACGATT CCGAATCCCG ACCTTCTACAGTTGCAGCAT 420 CGGGGAAAGT CCTTGTACCT GGAATCCAGA GTGATAGCTC CTCATCCTCAACAGACCAAT 480 TTGAGTTCAC TGAGACAGCT CCAGAAAATT CCCCAGCATC AACTGATGTGGATAGTTCAA 540 CAATGGAACA CGCTAGCCAG ATTAAAACTG AGAACGATGA CGTTGAGCCGTCAAGTGATC 600 TTACAGGAAG TGTTGAAGAG TTGGATTTTG CTTCATCACT ACAACTACAAGAAGGTGGTA 660 AACTGGAGGA GTCTAAAACA TTAAATACTT CTGAAGAGAC AATTATTGATGAATCTGATA 720 GGATCAGAGA GAGGGGCATC CCTCCACCTG GACTTGGTCA GAAGATTTATGAAATAGACC 780 CCCTTTTGAC AAACTATCGT CAACACCTTG ATTACAGGTA TTCACAGTACAAGAAAATGA 840 GGGAGGCAAT TGACAAGTAT GAGGGTGGTT TGGAAGCTTT TTCTCGTGGTTATGAAAAAA 900 TGGGTTTCAC TCGTAGTGCT ACAGGTATCA CTTACCGTGA GTGGGCTCCTGGTGCCCAGT 960 CAGCTGCTCT CATTGGAGAT TTCAACAATT GGGACGCAAA TGCTGACATTATGACTCGGA 1020 ATGAATTTGG TGTCTGGGAG ATTTTTCTGC CAAATAATGT GGATGGTTCTCCTGCAATTC 1080 CTCATGGGTC CAGAGTGAAG ATACGCATGG ACACTTCATC AGGTGTTAAGGATTCCATTC 1140 CTGCTTGGAT CAACTACTCT TTACAGCTTC CTGATGAAAT TCCATATAATGGAATATATT 1200 ATGATCCACC CGAAGAGGAG AGGTATGTCT TCCAACACCC ACGGCCAAAGAAACCAAAGT 1260 CGCTGAGAAT ATATGAATCT CATATTGGAA TGAGTAGTCC GGAGCCTAAAATTAACTCAT 1320 ACGTGAATTT TAGAGATGAA GTTCTTCCTC GCATAAAAAA CCTTGGGTACAATGCGGTGC 1380 AAATTATGGC TATTCAAGAG CATTCTTATT ATGCTAGTTT TGGTTATCATGTCACAAATT 1440 TTTTTGCACC AAGCAGCCGT TTTGGAACGC CCGACGACCT TAAGTCTTTGATTGATAAAG 1500 CTCATGAGCT AGGAATTGTT GTTCTCATGG ACATTGTTCA CAGCCATGCATCAAATAATA 1560 CTTTAGATGG ACTGAACATG TTTGACGGCA CAGATAGTTG TTACTTTCACTCTGGAGCTC 1620 GTGGTTATCA TTGGATGTGG GATTCCCGCC TCTTTAACTA TGGAAACTGGGAGGTACTTA 1680 GGTATCTTCT CTCAAATGCG AGATGGTGGT TGGATGAGTG CAAATTTGRTGGATTTAGAT 1740 TTGATGGTGT GACATCAATG ATGTATACTC ACCACGGATT ATCGGTGGGATTCACTGGGA 1800 ACTACGAGGA ATACTTTGGA CTCGCAACTG ATGTRGATGC TGCCGTGTATCTGATGCTGG 1860 CCAACGATCT TATTCATGGG CTTTTCCCAG ATGCAATTAC CATTGGTGAAGATGTTAGCG 1920 GAATGCCGAC ATTTTGTATT CCCGTTCAAG ATGGGGGTGT TGGCTTTGACTATCGGCTGC 1980 ATATGGCAAT TGCTGATAAA TGGATTGAGT TGCTCAAGAA ACGGGATGAGGATTGGAGAG 2040 TGGGTGATAT TGTTCATACA CTGACAAATA GAAGATGGTC GGAAAAGTGTGTTTCATACG 2100 CTGAAAGTCA TGATCAAGCT CTAGTCGGTG ATAAAACTAT AGCATTCTGGCTGATGGACA 2160 AGGATATGTA TGATTTTATG GCTTTGGATA GACCGTCAAC ATCATTAATAGATCGTGGGA 2220 TAGCATTGCA CAAGATGATT AGGCTTGTAA CTATGGGATT AGGAGGAGAAGGGTACCTAA 2280 ATTTCATGGG AAATGAATTC GGCCACCCTG AGTGGATTGA TTTCCCTAGGGCTGAACAAC 2340 ACCTCTCTGA TGGCTCAGTA ATTCCCGGAA ACCAATTCAG TTATGATAAATGCAGACGGA 2400 GATTTGACCT GGGAGATGCA GAATATTTAA GATACCGTGG GTTGCAAGAATTTGACCGGG 2460 CTATGCAGTA TCTTGAAGAT AAATATGAGT TTATGACTTC AGAACACCAGTTCATATCAC 2520 GAAAGGATGA AGGAGATAGG ATGATTGTAT TTGAAAAAGG AAACCTAGTTTTTGTCTTTA 2580 ATTTTCACTG GACAAAAAGC TATTCAGACT ATCGCATAGG CTGGCTGAAGCCTGGAAAAT 2640 ACAAGGTTGC CTTGGACTCA GATGATCCAC TTTTTGGTGG CTTCGGGAGAATTGATCATA 2700 ATGCCGAATG TTTCACCTTT GAAGGATGGT ATGATGATCG TCCTCGTTCAATTATGGTGT 2760 ATGCACCTAG TAGAACAGCA GTGGTCTATG CACTAGTAGA CAAAGAAGAAGAAGAAGAAG 2820 AAGTAGCAGT AGTAGAAGAA GTAGTAGTAG AAGAAGAATG AACGAACTTGTGATCGCGTT 2880 GAAAGATTTG AACGCTACAT AGAGCTTCTT GACGTATCTG GCAATATTGCATCAGTCTTG 2940 GCGGAATTTC ATGTGACAAA AGGTTTGCAA TTCTTTCCAC TATTAGTAGTGCAACGATAT 3000 ACGCAGAGAT GAAGTGCTGA ACAAACATAT GTAAAATCGA TGAATTTATGTCGAATGCTG 3060 GGACGGGCTT CAGCAGGTTT TGCTTAGTGA GTTCTGTAAA TTGTCATCTCTTTANATGTA 3120 CAGCCCACTA GAAATCAATT ATGTGAGACC TAAAAAACAA TAACCATAAAATGGAAATAG 3180 TGCTGATCTA ATGATGTTTT AANCCNNNNA AAAAAAAAAA AAAAACTCGA G3231 2578 base pairs nucleic acid single linear 19 TCATTAAAGA GGAGAAATTAACTATGAGAG GATCTCACCA TCACCATCAC CATGGGATCT 60 TGGCTGAAAA GTCTTCTTACAATTCCGAAT TCCGACCTTC TACAGTTGCA GCATCGGGGA 120 AAGTCCTTGT GCCTGGAACCCAGAGTGATA GCTCCTCATC CTCAACAAAC CAATTTGAGT 180 TCACTGAGAC ATCTCCAGAAAATTCCCCAG CATCAACTGA TGTAGATAGT TCAACAATGG 240 AACACGCTAG CCAGATTAAAACTGAGAACG ATGACGTTGA GCCGTCAAGT GATCTTACAG 300 GAAGTGTTGA AGAGCTGGATTTTGCTTCAT CACTACAACT ACAAGAAGGT GGTAAACTGG 360 AGGAGTCTAA AACATTAAATACTTCTGAAG AGACAATTAT TGATGAATCT GATAGGATCA 420 GAGAGAGGGG CATCCCTCCACCTGGACTTG GTCAGAAGAT TTATGAAATA GACCCCCTTT 480 TGACAAACTA TCGTCAACACCTTGATTACA GGTATTCACA GTACAAGAAA CTGAGGGAGG 540 CAATTGACAA GTATGAGGGTGGTTTGGAAG CTTTTTCTCG TGGTTATGAA AAAATGGGTT 600 TCACTCGTAG TGCTACAGGTATCACTTACC GTGAGTGGGC TCCTGGTGCC CAGTCAGCTG 660 CCCTCATTGG AGATTTCAACAATTGGGACG CAAATGCTGA CATTATGACT CGGAATGAAT 720 TTGGTGTCTG GGAGATTTTTCTGCCAAATA ATGTGGATGG TTCTCCTGCA ATTCCTCATG 780 GGTCCAGAGT GAAGATACGTATGGACACTC CATCAGGTGT TAAGGATTCC ATTCCTGCTT 840 GGATCAACTA CTCTTCACAGCTTCCTGATG AAATTCCATA TAATGGAATA TATTATGATC 900 CACCCGAAGA GGAGAGGTATATCTTCCAAC ACCCACGGCC AAAGAAACCA AAGTCGCTGA 960 GAATATATGA ATCTCATATTGGAATGAGTA GTCCGGAGCC TAAAATTAAC TCATACGTGA 1020 ATTTTAGAGA TGAAGTTCTTCCTCGCATAA AAAAGCTTGG GTACAATGCG GTGCAAATTA 1080 TGGCTATTCA AGAGCATTCTTATTATGCTA GTTTTGGTTA TCATGTCACA AATTTTTTTG 1140 CACCAAGCAG CCGTTTTGGAACGCCCGACG ACCTTAAGTC TTTGATTGAT AAAGCTCATG 1200 AGCTAGGAAT TGTTGTTCTCATGGACATTG TTCACAGCCA TGCATCAAAT AATACTTTAG 1260 ATGGACTGAA CATGTTTGACGGCACCGATA GTTGTTACTT TCACTCTGGA GCTCGTGGTT 1320 ATCATTGGAT GTGGGATTCCCGCCTTTTTA ACTATGGAAA CTGGGAGGTA CTTAGGTATC 1380 TTCTCTCAAA TGCGAGATGGTGGTTGGATG AGTTCAAATT TGATGGATTT AGATTTGATG 1440 GTGTGACATC AATGATGTATACTCACCACG GATTATCGGT GGGATTCACT GGGAACTACG 1500 AGGAATACTT TGGACTCGCAACTGATGTGG ATGCTGTTGT GTATCTGATG CTGGTCAACG 1560 ATCTTATTCA TGGGCTTTTCCCAGATGCAA TTACCATTGG TGAAGATGTT AGCGGAATGC 1620 CGACATTTTG TATTCCCGTTCAAGATGGGG GTGTTGGCTT TGACTATCGG CTGCATATGG 1680 CAATTGCTGA TAAATGGATTGAGTTGCTCA AGAAACGGGA TGAGGATTGG AGAGTGGGTG 1740 ATATTGTTCA TACACTGACAAATAGAAGAT GGTCGGAAAA GTGTGTTTCA TACGCTGAAA 1800 GTCATGATCA AGCTCTAGTCGGTGATAAAA CTATAGCATT CTGGCTGATG GACAAGGATA 1860 TGTATGATTT TATGGCTCTGGATAGACCGC CAACATCATT AATAGATCGT GGGATAGCAT 1920 TGCACAAGAT GATTAGGCTTGTAACTATGG GATTAGGAGG AGAAGGGTAC CTAAATTTCA 1980 TGGGAAATGA ATTCGGCCACCCTGAGTGGA TTGATTTCCC TAGGGCTGAA CAACACCTCT 2040 CTGATGACTC AGTAATTCCCGGAAACCAAT TCAGTTATGA TAAATGCAGA CGGAGATTTG 2100 ACCTGGGAGA TGCAGAATATTTAAGATACC GTGGGTTGCA AGAATTTGAC CGGGCTATGC 2160 AGTATCTTGA AGATAAATATGAGTTTATGA CTTCAGAACA CCAGTTCATA TCACGAAAGG 2220 ATGAAGGAGA TAGGATGATTGTATTTGAAA AAGGAAACCT AGTTTTTGTC TTTAATTTTC 2280 ACTGGACAAA AAGCTATTCAGACTATCGCA TAGGCTGCCT GAAGCCTGGA AAATACAAGG 2340 TTGCCTTGGA CTCAGATGATCCACTTTTTG GTGGCTTCGG GAGAATTGAT CATAATGCCG 2400 AATATTTCAC CTTTGAAGGATGGTATGATG ATCGTCCTCG TTCAATTATG GTGTATGCAC 2460 CTTGTAGAAC AGCAGTGGTCTATGCACTAG TAGACAAAGA AGAAGAAGAA GAAGAAGAAG 2520 AAGAAGAAGT AGCAGTAGTAGAAGAAGTAG TAGTAGAAGA AGAATGAACG AACTTGTG 2578 23 base pairs nucleicacid single linear 20 AATTTYATGG GNAAYGARTT YGG 23

We claim:
 1. A potato starch comprising amylose in an amount of at leastabout 35% to about 66%, as judged by the iodometric assay method ofMorrison & Laignelet, and amylopectin.
 2. A potato starch comprisingamylose in an amount of at least about 35%, as judged by the iodometricassay method of Morrison & Laignelet, wherein the starch forms asuspension in water at 10% w/w at about 40° C.
 3. The starch accordingto claim 1 or 2, having an amylose content of at least 37%.
 4. Thestarch according to claim 1 or 2, having an amylose content of at least40%.
 5. The starch according to claim 1 or 2, or having an amylosecontent of at least 50%.
 6. The starch according to claim 2, having anamylose content of about 66%.
 7. The starch according to claim 2, havingan amylose content of about 35%-about 66%.
 8. The starch according toclaim 1 or 2, which as extracted from a potato plant by wet milling atambient temperature has a viscosity onset temperature in the range70-95° C., as judged by viscoamylograph of a 10% w/w aqueous suspensionthereof, performed at atmospheric pressure using the Newport ScientificRapid Visco Analyser 3C with a heating profile of holding at 50° C. for2 minutes, heating from 50 to 95° C. at a rate of 1.5° C. per minute,holding at 95° C. for 15 minutes, cooling from 95 to 50° C. at a rate of1.5° C. per minute, and then holding at 50° C. for 15 minutes.
 9. Thestarch according to claim 1 or 2, which as extracted from a potato plantby wet milling at ambient temperature has peak viscosity in the range500-12 stirring number units (SNUs), as judged by viscoamylograph of a10% w/w aqueous suspension thereof, performed at atmospheric pressureusing the Newport Scientific Rapid Visco Analyser 3C with a beatingprofile of holding at 50° C. for 2 minutes, heating from 50 to 95° C. ata rate of 1.5° C. per minute, holding at 95° C. for 15 minutes, coolingfrom 95 to 50° C. at a rate of 1.5° C. per minute, and then holding at50° C. for 15 minutes.
 10. The starch according to claim 1 or 2, whichas extracted from a potato plant by wet milling at ambient temperaturehas a pasting viscosity in the range 214-434 SNUs, as judged byviscoamylograph of a 10% w/w aqueous suspension thereof, performed atatmospheric pressure using the Newport Scientific Rapid Visco Analyser3C with a heating profile of holding at 50° C. for 2 minutes, heatingfrom 50 to 95° C. at a rate of 1.5° C. per minute, holding at 95° C. for15 minutes, cooling from 95 to 50° C. at a rate or 1.5° C. per minute,and then holding at 50° C. for 15 minutes.
 11. The starch according toclaim 1 or 2, which as extracted from a potato plant by wet milling atambient temperature has a set-back viscosity in the range 450-618 SNUs,as judged by viscoamylograph of a 10% ww aqueous suspension thereof,performed at atmospheric pressure using the Newport Scientific RapidVisco Analyser 3C with a beating profile of holding at 50° C. for 2minutes, heating from 50 to 95° C. at a rate of 1.5° C. per minute,holding at 95° C. for 15 minutes, cooling from 95 to 50° C. at a rate of1.5° C. per minute, and then holding at 50° C. for 15 minutes.
 12. Thestarch according to claim 1 or 2, which as extracted from a potato plantby wet milling at ambient temperature has a peak viscosity in the range14-192 SNUs, as judged by viscoamylograph of a 10% w/w aqueoussuspension thereof, performed at atmospheric pressure using the New PortScientific Rapid Visco Analyser 3C with a heating profile of holding at50° C. for 2 minutes, heating from 50 to 95° C. at a rate of 1.5° C. perminute, holding at 95° C. for 15 minutes, cooling from 95 to 50° C. at arate of 1.5° C. per minute, and then holding at 50° C. for 15 minutes.13. The starch according to claim 1 or 2, which as extracted from apotato plant by wet milling at ambient temperature has a peak viscosityin the range 200-500 SNUs and a set-back viscosity in the range 275-618SNUs as judged by viscoamylograph of a 10% w/w aqueous suspensionthereof, performed at atmospheric pressure using the Newport ScientificRapid Visco Analyser 3C with a heating profile of holding at 50° C. for2 minutes, beating from 50 to 95° C. at a rate of 1.5° C. per minute,holding at 95° C. for 15 minutes, cooling from 95 to 50° C. at a rate of1.5° C. per minute, and then holding at 50° C. for 15 minutes.
 14. Thestarch according to claim 1 or 2, or which as extracted from a potatoplant by wet milling at ambient temperature has a viscosity which doesnot decrease between the start of the heating phase (step 2) and thestart of the final holding phase (step 5) and has a set-back viscosityof 303 SNUs or less as judged by viscoamylograph of a 10% w/w aqueoussuspension thereof, performed at atmospheric pressure using the NewportScientific Rapid Visco Analyser 3C with a heating profile of holding at50° C. for 2 minutes, heating from 50 to 95° C. at a rate of 1.5° C. perminute, holding at 95° C. for 15 minutes, cooling from 95 to 50° C. at arate of 1.5° C. per minute, and then holding at 50° C. for 15 minutes.15. The starch according to claim 1 or 2, which as extracted from apotato plant by wet milling at ambient temperature displays nosignificant increase in viscosity as judged by viscoamylograph conductedof a 10% w/w aqueous suspension thereof, performed at atmosphericpressure using the Newport Scientific Rapid Visco Analyser 3C with aheating profile of holding at 50° C. for 2 minutes, heating from 50 to95° C. at a rate of 1.5° C. per minute, holding at 95° C. for 15minutes, cooling from 95 to 50° C. at a rate of 1.5° C. per minute, andthen holding at 50° C. for 15 minutes.
 16. The starch according to claim1 or 2, which as extracted from a potato plant, has a phosphorus contentin excess of 200 mg/100 grams dry weight starch.
 17. The starchaccording to claim 16, having a phosphorus content in the range 200-240mg/100 grams dry weight starch.
 18. The starch according to claim 1 or2, wherein the starch has been further treated physically, chemically,and/or enzynmatically.
 19. The starch according to claim 18, wherein thestarch is a resistant starch.
 20. The starch according to claim 19,wherein the starch has in excess of 5% total dietary fiber, asdetermined according to the Protsky method.
 21. A potato starchobtainable from a plant having characteristics altered by a methodselected from the group consisting of (a) introducing into the plant aportion of a nucleotide sequence encoding an effective portion of aclass A starch branching enzyme (SBE) obtainable from potato plants tocomplement the branching enzyme mutation in E coli KV 832 cells andwhich is active when expressed in E. coli in the phosphorylationstimulation assay operably linked to a suitable promoter active in theplant so as to affect the expression of a gene present in plant; (b)introducing into the plant a portion of a nucleotide sequence encodingan effective portion of a class A starch branching enzyme (SBE)obtainable from potato plants operably linked to a suitable promoteractive in the plant so as to affect the expression of a gene present inthe plant, wherein the nucleotide sequence is operably linked in theanti-sense orientation to a suitable promoter active in the plant; (c)introducing into the plant a portion of a nucleotide sequence encodingan effective portion of a class A starch branching enzyme (SBE)obtainable from potato plants operably linked to a suitable promoteractive in the plant so as to affect the expression of a gene present inthe plant, wherein the introduced sequence comprises at least one regionselected from the group consisting of a 5′ untranslated region, a 3′untranslated region, and a coding region of the potato SBE class A SBEoperably linked in the sense orientation to a promoter active in theplant; (d) introducing into the plant a portion of a nucleotide sequenceencoding an effective portion of a class A starch branching enzyme (SBE)obtainable from potato plants operably linked to a suitable promoteractive in the plant so as to affect the expression of a gene present inthe plant further comprising introducing into the plant one or morefurther sequences; (e) introducing into the plant a portion of anucleotide sequence encoding an effective portion of a class A starchbranching enzyme (SBE) obtainable from potato plants operably linked toa suitable promoter active in the plant so as to affect the expressionof a gene present in the plant further comprising introducing into theplant one or more further sequences operably linked in the anti-senseorientation to a suitable promoter active in the plant; and (f)introducing into the plant a portion of a nucleotide sequence encodingan effective portion of a class A starch branching enzyme (SBE)obtainable from potato plants operably linked to a suitable promoteractive in the plant so as to affect the expression of a gene present inthe plant further comprising introducing into the plant a portion of aclass B SBE nucleotide sequence wherein the portion is effective tocomplement the branching enzyme mutation in E. coli KV 832 cells andwhich is active when expressed in E. coli in the phosphorylationstimulation assay, and the starch has an amylose content of at least 35%as judged by the iodometric assay method of Morrison & Laignelet,wherein the starch forms a suspension in water at 10% w/w at about 40°C.
 22. A potato starch obtainable from a plant having characteristicsaltered by a method selected from the group consisting of (a)introducing into the plant a portion of a nucleotide sequence encodingan effective portion of a class A starch branching enzyme (SBE)obtainable from potato plants to complement the branching enzymemutation in E coli KV 832 cells and which is active when expressed in E.coli in the phosphorylation stimulation assay operably linked to asuitable promoter active in the plant so as to affect the expression ofa gene present in the plant; (b) introducing into the plant a portion ofa nucleotide sequence encoding an effective portion of a class A starchbranching enzyme (SBE) obtainable from potato plants operably linked toa suitable promoter active in the plant so as to affect the expressionof a gene present in the plant, wherein the nucleotide sequence isoperably linked in the anti-sense orientation to a suitable promoteractive in the plant; (c) introducing into the plant a portion of anucleotide sequence encoding an effective portion of a class A starchbranching enzyme (SBE) obtainable from potato plants operably linked toa suitable promoter active in the plant so as to affect the expressionof a gene present in the plant, wherein the introduced sequencecomprises at least one region selected from the group consisting of a 5′untranslated region, a 3′ untranslated region, and a coding region ofthe potato SBE class A SBE operably linked in the sense orientation to apromoter active in the plant; (d) introducing into the plant a portionof nucleotide sequence encoding an effective portion of a class A starchbranching enzyme (SBE) obtainable from potato plants operably linked toa suitable promoter active in the plant so as to affect the expressionof a gene present in the plant further comprising introducing into theplant one or more further sequences; (e) introducing into the plant aportion of a nucleotide sequence encoding an effective portion of aclass A starch branching enzyme (SBE) obtainable front potato plantsoperably linked to a suitable promoter active in the plant so as theaffect the expression of a gene present in the plant further comprisingintroducing into the plant one or more further sequences operably linkedin the anti-sense orientation to a suitable promoter active in theplant; and (f) introducing into the plant a portion of a nucleotidesequence encoding an effective portion of a class A starch branchingenzyme (SBE) obtainable from potato plants operably linked to a suitablepromoter active in the plant so as to affect the expression of a genepresent in the plant further comprising introducing into the plant aportion of a class B SBE nucleotide sequence wherein the portion iseffective to complement the branching enzyme mutation in E. coli KV 832cells and which is active when expressed in E. coli in thephosphorylation stimulation assay, and the starch has an amylose contentof at least about 35% to about 66% as judged by the iodometric assaymethod of Morrison & Laignelet.
 23. A method of modifying starch invitro, comprising treating starch with an effective amount of a class Astarch branching enzyme (SBE) polypeptide obtainable from potato plantsand encoded by a nucleotide sequence encoding an effective portion of aclass A SBE obtainable from potato plants, wherein the amount iseffective to complement the branching enzyme mutation in E coli KV 832cells and which is active when expressed in E. coli in thephosphorylation stimulation assay.